Microfluidic systems for epidermal sampling and sensing

ABSTRACT

Provided are microfluidic systems for monitoring a biofluid property and related methods. Specially configured microfluidic networks and associated structural support and functional elements, including flexible substrates, capping layers, and fluidic conduits and controllers, provide reliable biofluid collection. Optical components and indicators provide a reliable and readily observable readout, including of any of a number of biofluid properties, including directly from biofluid collected in the microfluidic network.

This application claims the benefit of U.S. Provisional PatentApplication Nos. 62/514,489, 62/514,515, 62/514,374, 62/514,455,62/514,520, 62/514,468, 62/514,546, 62/514,559, and 62/514,436, allfiled Jun. 2, 2017, each of which is specifically incorporated byreference to the extent not inconsistent herewith.

BACKGROUND OF INVENTION

Microfluidics provides a versatile technology platform affecting a widerange of industries and commercial products. In the field of medicaldiagnostics, for example, microfluidics has been essential to thedevelopment of entirely new classes of sensors and assays with potentialfor revolutionizing medical diagnosis and the treatment of disease. Labon a chip and microarray systems, for example, have been developed forclinical pathology taking advantage of microfluidic sample collection,preparation and handling to achieve highly sensitivity and rapid pointof care analysis of biomarkers in minute quantities of biofluid. Theadvances in microfluidics have also been leveraged to support otherbiotechnology and medical applications including high throughput DNAsequencing, mass spectrometry-based proteomics, cellular expression andimaging.

Wearable systems are another technology for which advances inmicrofluidics has potential to enable new classes of products andadvanced modes of functionality. Recent developments in epidermalelectronics, for example, provide a class of skin-mounted sensors andactuators compatible with efficient microfluidic sampling at theinterface of the skin. Such microfluidics-enabled epidermal systems havepotential to support a broad range of clinical applications inhealthcare including analysis of biomarkers, drug administration, andreal time diagnosis and monitoring of medical conditions includingdiabetes, inflammation and hydration state. Examples include,US20060253011; US20100179403; WO 2016/025468; WO 2016/025438;WO2010030609; US20070027383; US20070179371A1; U.S. Pat. Nos. 4,960,467;6,198,953; and WO2009025698A1.

As will be understood from the forgoing, the development of wearablesystems is needed in a manner that integrates microfluidic functionalitywith tissue mounted sensing and actuation. Wearable systems havingphysical formats and mechanical properties that provide a robustinterface with the skin to achieve quantitatively reliable collectionand handling of biofluids over clinically (and commercially) relevanttime intervals are needed. In addition, microfluidic systems are neededthat are capable of effective collection, pretreatment, storage andanalysis of biofluids to support a range of applications for wearablesystems, including for physical exertion applications, medicaldiagnostics and therapy, and general well-being.

There is a particular need for reliable biofluid collection, retention,and monitoring/analysis under a range of extreme environmentalconditions, including in wet environments, dry environments, hot/coldtemperatures, active/passive users, healthy/unwell users. Proved hereinare systems that address these needs using specially configuredmicrofluidic networks and associated components for desired fluidiccollection and pathways depending on the application of interest.

SUMMARY OF THE INVENTION

Provided are microfluidic systems and methods for the measurement andcharacterization of biofluids in a manner that is versatile and that canbe tailored to a wide range of applications. For example, the systemsare compatible for monitoring a biofluid property in different flowregimes, including low flow and high flow regimes, where biofluids arecorrespondingly available in small quantities or in larger quantities.This can be achieved by providing a plurality of microfluidic networks,with each network tailored to a specific flow regime. One manner inwhich this can be achieved is by adjusting microfluidic geometry andsize and fluid control elements within the microfluidic networks. Forexample, flow path can be further controlled with valves that open whena desired flow condition is satisfied, such as a plurality of capillaryburst valves having specially selected and unique burst valve pressuresso that controlled biofluid flow is achieved in the various microfluidicnetworks.

The microfluidic system may comprise: a flexible substrate; and at leasttwo microfluidic networks, each microfluidic network configured toindependently monitor a biofluid property; wherein each microfluidicnetwork comprises: a microfluidic inlet conduit network at leastpartially embedded in or supported by the substrate; a biofluid inletfluidically connected with the microfluidic inlet conduit network tointroduce a biofluid from the skin surface to the microfluidic inletconduit during use; a plurality of reservoir chambers, each reservoirchamber fluidically connected with the microfluidic inlet conduitnetwork; a plurality of capillary burst valves fluidically connectedwith the microfluidic conduit network, each capillary burst valvepositioned between fluidically adjacent reservoir chambers; and aplurality of colorimetric sensors, each positioned in a unique reservoirchamber to monitor the biofluid property.

The at least two microfluidic networks may differ from each other by (i)a biofluid inlet dimension, (ii) a reservoir chamber volume of each ofthe plurality of reservoir chambers, (iii) a burst pressure of each ofthe plurality of capillary burst valves, or (iv) any combinationthereof.

The microfluidic system may comprise: a plurality of reservoir networksat least partially embedded in or supported by a flexible substrate,each reservoir network comprising: a reservoir chamber; a biofluid inletfluidically connected to the reservoir chamber via a capillary burstvalve, having a burst pressure, to introduce a biofluid from a skinsurface to the reservoir chamber; an outlet fluidically connected to areservoir chamber.

Also provided are methods for measuring a biofluid property for abiofluid released from skin using any of the microfluidic systemsdisclosed herein.

Any of the systems described herein may utilize a means for minimizingbiofluid loss and/or increasing biofluid collection efficiency. Forexample, any of the systems described herein may utilize a capping layerto mitigate unwanted biofluid loss (or entry), to thereby provideimproved device performance, reliability and accuracy. Any of thesystems and methods described herein may contain a biofluid gellingagent in the microfluidic network, wherein conversion of a fluid into agel within the microfluidic network decreases unwanted biofluid loss.Any of the systems and methods described herein may contain an absorbentin the microfluidic network, wherein the biofluid is at least partiallyabsorbed by the absorbent, thereby minimizing biofluid loss. Any of thegelling agents and/or absorbents may be positioned in a reservoirchamber, or at specific locations in the network, depending on theapplication of interest and/or surrounding environmental conditions.

To facilitate reliable and efficient measurement, any of the systems andmethods may utilize specially patterned elements, including patternedgrating in a portion of the microfluidic network, to provide controlledchange in an optical transmission property. In this manner, transmissionof incident electromagnetic radiation through the patterned gratingchanges as a function of the amount of the biofluid in the microfluidicchannel or reservoir chamber. An indicator may be provided in opticalcommunication with the patterned grating, wherein changes in thetransmission of incident electromagnetic radiation through the gratingchanges the appearance of the indicator.

The provided systems and methods utilize capillary force and/or heatsources to help drive biofluid generation and/or capture, which wouldotherwise be difficult to measure and/or characterize. The heat sourcesmay be intrinsic to the system, such a heater. The heat sources may beextrinsic to the system, such as by hot water, generated by a shower orbath, for example, to generate biofluid release from a skin surface. Anabsorbent may be used in the microfluidic network to generate a biofluidcollection force in a manner that draws out biofluid from the skinsurface to the network for collection and analysis.

The systems and methods may be characterized as providing a platform forchrono-sampling, including for various fluid flow regimes andconstituents therein, such as biomarkers. This may be achieved usingmultiple microfluidic networks. In this manner, the microfluidic systemfor monitoring a biofluid property may comprise: a flexible substrate;at least two microfluidic networks, each microfluidic network configuredto independently monitor a biofluid property; wherein each microfluidicnetwork comprises: a microfluidic inlet conduit network at leastpartially embedded in or supported by the substrate; a biofluid inletfluidically connected with the microfluidic inlet conduit network tointroduce a biofluid from a skin surface to the microfluidic inletconduit during use; a plurality of reservoir chambers, each reservoirchamber fluidically connected with the microfluidic inlet conduitnetwork; and a plurality of capillary burst valves fluidically connectedwith the microfluidic conduit network, each capillary burst valvepositioned between fluidically adjacent reservoir chambers.

Any of the microfluidic systems may comprise a plurality of colorimetricsensors, wherein each colorimetric sensor is positioned in a uniquereservoir chamber to monitor the biofluid property.

Any of the microfluidic systems may have at least two microfluidicnetworks that differ from each other by: (i) a biofluid inlet dimension,(ii) a reservoir chamber volume of each of the plurality of reservoirchambers, (iii) a burst pressure of each of the plurality of capillaryburst valves, (iv) a chemical composition of a chemically-mediatedreaction chamber, or (v) any combination thereof.

Any of the microfluidic systems may have a first microfluidic networkconfigured to monitor a biofluid parameter associated with a low-flowbiofluid regime and a second microfluidic network is configured tomonitor a biofluid parameter associated with a high-flow biofluidregime, and the biofluid property is biofluid amount; biofluid analyteconcentration; biomarker presence or absence; or a combination thereof.In this manner, a single system can accommodate large changes inbiofluid generation, such as sweating in a hot, humid environment duringstrenuous exercise (corresponding to high sweat-rate) versus sedentaryactivity in a cool climate (very little sweat generated). As discussedherein below, additional components may be included to help drivebiofluid collection, particularly in applications where no to littlesweat is generated.

Sizing and geometry of inlets and microchannels can be selected tocorrespond to a desired flow range. In this aspect, various flow regimesare accommodated. For example, the microfluidic system may have a highbiofluid loss regime that is at least 10 times greater than the lowbiofluid loss regime. By varying fluidic conduit sizes, flow resistanceis effectively varied, thereby controlling flow-rate (Q=ΔP/R).Similarly, use of capillary burst valves with selected burst-valvepressures may also be used to control biofluid introduction to differentmicrofluidic networks.

Each microfluidic network may further comprise: at least onemicrofluidic outlet conduit, each microfluidic outlet conduitfluidically connected to at least one of the plurality of reservoirchambers and configured to relieve gas back pressure from themicrofluidic inlet conduit network.

Any of the microfluidic systems may be described as having the pluralityof reservoir chambers chemically decoupled from each other forindependent biofluid property detection and/or time sequential biofluidproperty monitoring. This chemical decoupling may be achieved byselection of microchannel dimensions (e.g., length and width),particularly between fluidically adjacent reservoir chambers. Forexample, by ensuring Reynold's number is in the laminar range, includingless than 100, or less than 10, or less than 1, mixing is minimized, anddiffusion reduced for sufficiently long distances between adjacentreservoir chambers so that diffusion between the chambers is unlikelyover the relevant time-scale.

Any of the microfluidic systems may further comprise a plurality ofcapillary burst valves, wherein at least one capillary burst valve ispositioned between fluidically adjacent reservoir chambers. In thismanner, pressure may be determined by visual observation of filling ofreservoir chambers, with higher pressures associated with differentreservoir filling.

Any of the systems may measure pressure of a biofluid, such as thepressure associated with one or more sweat glands. The microfluidicsystem for measuring a biofluid pressure may comprise: a flexiblesubstrate; a plurality of reservoir networks at least partially embeddedin or supported by the flexible substrate, wherein each reservoirnetwork comprises: a reservoir chamber; a biofluid inlet fluidicallyconnected to the reservoir chamber via a capillary burst valve tointroduce a biofluid from a skin surface to the reservoir chamber,wherein the capillary burst valve has a burst pressure; and an outletfluidically connected to the reservoir chamber; wherein the burstpressure of each capillary burst valve is selected to correspond to apressure range of the biofluid from the skin surface.

At least one of the plurality of reservoir networks (e.g., reservoirchamber) may have a unique capillary burst valve pressure, therebyproviding a unique pressure measure associated with the correspondingreservoir network. In this manner, any number of distinct pressures maybe measured.

The biofluid inlet may be fluidically aligned with a biofluid source ofthe skin surface during use.

Any of the reservoir networks (chambers) may further comprise at leastone colorimetric sensor to provide an optical readout.

At least a portion of the capillary burst valves may be fluidicallyaligned in a serial configuration and have a burst valve pressure thatincreases and spans a minimum to maximum pressure, such as a range thatis greater than 0 kPa and less than 10 kPa, and any subranges thereof.

Any of the systems may have specific colorimetric sensor, such as asensor comprising silver chloranilate. The microfluidic system tomeasure a biofluid property, may comprise: a flexible substrate; amicrofluidic inlet conduit network at least partially embedded in orsupported by the flexible substrate; a biofluid inlet fluidicallyconnected to the microfluidic inlet conduit network to introduce abiofluid from the skin surface to the microfluidic inlet conduit duringuse; a plurality of reservoir chambers, each reservoir chamberfluidically connected with the microfluidic inlet conduit network; aplurality of capillary burst valves fluidically connected with themicrofluidic conduit network, each capillary burst valve positionedbetween fluidically adjacent reservoir chambers; a microfluidic outletconduit network fluidically connected with the plurality of reservoirchambers and configured to relieve gas back pressure from themicrofluidic inlet conduit network; a plurality of colorimetric sensors,each positioned in a unique reservoir chamber to measure a biofluidproperty; and wherein: at least one of the colorimetric sensors has acolor-responsive reagent to measure chloride in the biofluid. Thecolor-responsive reagent may comprise silver chloranilate.

The microfluidic system may further comprise a color indicator strippositioned between any two fluidically adjacent reservoir chambers.

Any of the microfluidic systems may further comprise a capping layerconnected to a skin-facing surface and/or a back-facing surface of theflexible substrate.

The microfluidic system may comprise: a flexible substrate having askin-facing surface and a back-facing surface; a microfluidic network atleast partially embedded in or supported by the flexible substrate; asensor fluidically connected to the microfluidic network, wherein themicrofluidic network is configured to transport a biofluid from a skinsurface to the sensor; and a capping layer, having a capping layerskin-facing surface and a back-facing surface, wherein the back-facingsurface of the capping layer is attached to the skin-facing surface ofthe substrate; wherein the flexible substrate is at least partiallyformed of a thermoplastic elastomer or a polymer configured to provide ahigh barrier to vapor or liquid water transmission.

The capping layer may be at least partially formed of a thermoplasticelastomer and an additive. The flexible substrate and the capping layermay be formed of a common thermoplastic elastomer composition. Theflexible substrate and the capping layer may have a common additive.

Examples of thermoplastic elastomers include a styrene copolymer, suchas selected from the group consisting ofstyrene-ethylene-butadiene-styrene (SEBS), styrene-isoprene-styrene(SIS), styrene-butadiene-styrene (SBS), and any combination thereof.

The thermoplastic elastomer may have a weight fraction of styrenecopolymer selected from the range of 10% to 50%.

The additive may be a hydrocarbon compound characterized by a molecularweight less than a user-selected molecular weight, such as a molecularweight that is less than 1000 g/mol. The additive may be paraffin oil.

The thermoplastic elastomer may have a weight ratio of additive tostyrene copolymer selected from the range of 1 to 3.

Any of the microfluidic systems may have a capping layer that comprisesa spatially distributed pattern of relief, recess, or relief and recessfeatures to achieve a desired mechanical property while maintaining highbarrier to water vapor or liquid transmission. The pattern may comprisea symmetrical pattern. The pattern may be selected to achieve a desiredmechanical property of flexibility and stretchability of the cappinglayer that is substantially matched to the flexible substrate. Forexample, the mechanical property may be a Young's modulus of less than100 MPa, a net bending stiffness of less than 1 nN m, and/or a thicknessof less than 5 mm.

The spatially distributed pattern may be spatially aligned with at leasta portion of the microfluidic network. For example, recess features,including passages, may be aligned with inlets to facilitate biofluidflow to the microfluidic inlets.

An of the capping layers described herein may be at least partiallyformed of a rigid polymer selected from the group consisting of apolyolefin, a polyester, a fluorocarbon, a polyamide, a polyimide, andany combination thereof. The polyolefin may be selected from the groupconsisting of polyethylene, polypropylene and polyisobutylene; thepolyester is selected from the group consisting of polyethyleneterephthalate and polyethylene naphthalate; the fluorocarbon is selectedfrom the group consisting of polyvinylidene chloride andpolytetrafluoroethylene; the polyamide is a nylon; and/or the polyimideis a poly-oxydiphenylene-pyromellitimide.

Any of the microfluidic systems may further comprise an adhesive layeron the skin facing surface of the capping layer; wherein the adhesivelayer comprises an adhesive compound capable of reversibly adhering thesystem to the skin surface. For systems that do not have a cappinglayer, the adhesive layer may be positioned on a skin-facing surface ofthe flexible substrate. The adhesive layer may comprise medical-gradeacrylic.

The substrate, the capping layer, the adhesive compound, or anycombination thereof may further comprise a tackifier additive. Thesubstrate, the capping layer, or both the substrate and the cappinglayer may have a weight fraction of tackifier additive of between 30% to80%. The tackifier additive may be rosin gum.

Any of the microfluidic systems may have a microfluidic networkcomprising a plurality of reservoirs and a microfluidic inlet conduitnetwork having a biofluid inlet to introduce the biofluid to themicrofluidic network; and wherein the microfluidic outlet conduitnetwork is fluidically connected to the plurality of reservoirs. Themicrofluidic network may further comprise a microfluidic outlet conduitnetwork fluidically connected to the plurality of reservoirs, themicrofluidic inlet conduit network, and an outlet, and wherein theoutlet is configured to (i) provide for release of gas back pressurefrom the microfluidic inlet conduit network, and (ii) prevent ingress ofa liquid from a surrounding environment into the microfluidic outletconduit network.

Any of the microfluidic systems may have a sensor that is a colorimetricsensor. Any of the microfluidic systems may have a sensor that is anelectrochemical sensor. Any of the microfluidic systems may comprise twoor more sensors, including at least one colorimetric sensor and oneelectrochemical sensor.

The colorimetric sensor may be positioned in one of the plurality ofreservoirs. The electrochemical sensor may be positioned in one of theplurality of reservoirs.

Any of the microfluidic systems may further comprise a biofluid gellingadditive or an absorbent contained within the microfluidic network.

Any of the microfluidic systems may have a biofluid gelling additivecomprising two or more unique biofluid gelling additives.

The biofluid gelling agent may be configured to mix or react with thebiofluid to increase a biofluid viscosity. The increase in biofluidviscosity may be by at least a factor of 2 of the biofluid viscositybefore mixing or reacting with the biofluid gelling agent. In thismanner, risk of leakage may be reduced, including through one or more ofthe CBV's. The biofluid gelling agent may comprise cellulose or aderivative thereof. The biofluid gelling agent may be methyl celluloseor hydroxypropyl methylcellulose.

The weight ratio of the biofluid gelling agent to biofluid, in at leastone of the plurality of reservoirs, may be selected from the range of0.1 to 1, or any subranges thereof.

An of the flexible substrates may be a functional substrate.

Also provided herein are microfluidic systems configured to minimizeunwanted fluid loss from the system, such as to the surroundingenvironment or skin surface, including by a biofluid or an absorbent.Accordingly, the microfluidic system, may comprise: a flexiblesubstrate; a microfluidic network at least partially embedded in orsupported by the flexible substrate; a sensor fluidically connected tothe microfluidic network, wherein the microfluidic network is configuredtransport a biofluid from a skin surface to the sensor; and a biofluidgelling additive or a biofluid absorbent contained in the microfluidicnetwork to reduce biofluid loss from the microfluidic network.

The microfluidic network may comprise: a plurality of reservoirs; abiofluid inlet to introduce a biofluid to the microfluidic network; anda microfluidic inlet conduit network fluidically connected to thebiofluid inlet and the plurality of reservoirs to introduce a biofluidto the reservoirs.

The microfluidic network may further comprise: a microfluidic outletconduit network fluidically connected to the plurality of reservoirs;and an outlet fluidically connected to the microfluidic outlet conduit.The outlet is configured to: provide for release of gas back pressurefrom the microfluidic inlet conduit network, and prevent ingress of aliquid from an environment surrounding the system into the microfluidicoutlet conduit network.

The sensor may be a colorimetric sensor or an electrochemical sensor.The sensor may be positioned in one of the plurality of reservoirs.

The microfluidic system may comprise two or more sensors, including forsensing different biofluid properties.

For microfluidic systems having a biofluid gelling additive, thebiofluid gelling additive may be positioned in at least one of theplurality of reservoirs.

The microfluidic system may comprise two or more biofluid gellingadditives.

The biofluid gelling agent may be configured to mix or react with thebiofluid to increase a biofluid viscosity. The increase in biofluidviscosity may be by at least a factor of 2 of the biofluid viscositybefore mixing or reacting with the biofluid gelling agent.

The biofluid gelling agent may be at least partially formed of celluloseor a derivative thereof, such as methyl cellulose or hydroxypropylmethylcellulose.

The weight ratio of the biofluid gelling agent to biofluid, in at leastone of the plurality of reservoirs, may be selected from the range of0.1 to 1.

Any of the microfluidic systems may further comprise a capping layer,having a capping layer skin facing surface and a back surface, whereinthe back surface is affixed to a skin facing surface of the substrate.

The flexible substrate and/or capping layer may be at least partiallyformed of a thermoplastic elastomer having an additive. The substrateand the capping layer may have a common thermoplastic elastomercomposition, or a different thermoplastic elastomer composition. Thesubstrate and the capping layer may have a common additive.

The thermoplastic elastomer may be a styrene copolymer selected from thegroup consisting of styrene-ethylene-butadiene-styrene (SEBS),styrene-isoprene-styrene (SIS), styrene-butadiene-styrene (SBS), and anycombination thereof. The thermoplastic elastomer may have a weightfraction of styrene copolymer selected from the range of 10% to 50%

The additive may be a hydrocarbon compound characterized by a molecularweight less than a user-selected molecular weight. The additive may beparaffin oil.

The thermoplastic elastomer may have a weight ratio of additive tostyrene copolymer selected from the range of 1 to 3.

Any of the microfluidic systems may have a capping layer comprising aspatially distributed pattern of relief, recess, or relief and recessfeatures to achieve a desired mechanical property while maintaining highbarrier to water vapor or liquid transmission. The pattern may comprisea symmetrical pattern. The pattern may be selected to achieve a desiredmechanical property of flexibility and stretchability of the cappinglayer that is substantially matched to the flexible substrate, whereinthe mechanical property is one or more of a Young's modulus, a bendingstiffness or an average thickness. In this aspect, substantially matchedrefers to a bulk property that is within 30% of the bulk property of theflexible substrate.

The pattern may be spatially aligned with at least a portion of themicrofluidic network.

Any of the microfluidic systems may further comprise an expunge portfluidically connected with the reservoir chamber for the removal ofbiofluid from the reservoir chamber. In this manner, the microfluidicsystem, specifically the microfluidic network, may be reused.

Any of the microfluidic systems may further comprise optical componentsto facilitate detection of a biofluid property or characteristic.Examples of optical components include diffusors, lenses, diffractiongrates and the like. An epidermally-mountable microfluidic system formeasuring a characteristic of a biofluid from a skin surface maycomprise: a flexible substrate; a biofluid inlet embedded on orsupported by the substrate for receiving the biofluid from the skinsurface; and a microfluidic channel fluidically connected to thebiofluid inlet for receiving at least a portion of a biofluid from thebiofluid inlet, the microfluidic channel having a patterned grating. Inthis manner, transmission of incident electromagnetic radiation throughthe patterned grating changes as a function of a biofluid amount in themicrofluidic channel. The grating, of course, may be positioned in othercomponents of the network, such as in a reservoir chamber.

The system may further comprise an indicator in optical communicationthe patterned grating; wherein changes in the transmission of incidentelectromagnetic radiation through the grating changes the appearance ofthe indicator.

The patterned grating may comprise a hydrophilic polymer; and whereintransmission of the incident electromagnetic radiation by the patternedgrating increases when the chamber is filled with the biofluid.

The patterned grating may comprise a hydrophobic polymer; and whereintransmission of the incident electromagnetic radiation by the patternedgrating decreases when the chamber is filled with the biofluid.

The system may further comprise an expunge port fluidically connectedwith the reservoir chamber for the removal of the biofluid from thereservoir chamber.

The system may further comprise an adhesive layer, such as an adhesivecapable of reversibly adhering to the skin surface.

The adhesive layer may comprise medical grade acrylic or medical gradesilicone.

The expunge port may comprise two outlets.

Any of the systems may further comprise a capillary burst valvefluidically connected to said expunge port and said reservoir chamber.The capillary burst valve may be positioned between said expunge portand said reservoir chamber.

The system may have a patterned grating that is nanopatterned ormicropatterned.

The system may be configured to measure otherwise insensible sweat loss,including by the use of absorbents that assist in driving biofluid intothe network. For example, the epidermal microfluidic system formeasuring a characteristic of a biofluid; may comprise: a flexiblesubstrate; a collection layer embedded in or supported by the flexiblesubstrate, wherein the collection layer promotes transport of thebiofluid from the skin surface; at least one reservoir chamber embeddedin or supported by the flexible substrate and fluidically connected tothe collection layer, the reservoir chamber having: an absorbentprovided to receive at least a portion of the biofluid from thecollection layer; and a sensor for measuring a characteristic of thebiofluid received by the absorbent; wherein the absorbent provides aforce for transporting the biofluid that is greater than a capillaryforce of the collection layer for transporting the biofluid.

The epidermal microfluidic system for measuring a characteristic of abiofluid, may comprise: a flexible substrate; a radiofrequency (RF)heater embedded in or supported by the flexible substrate; wherein theRF heater is capable of increasing a temperature of the skin surface,thereby increasing the release rate of the biofluid; and at least onesensor embedded in or supported by the flexible substrate to measure thecharacteristic of the biofluid.

The biofluid characteristic may be the amount of sweat loss or presenceor absence of a biomarker contained in a biofluid released from a skinsurface, such as a gland, a wound, or the like.

The sensor may be an electronic sensor. The electronic sensor maycomprise one or more high sensitivity electrodes configured to measure achange in an electrical parameter caused by biofluid received by theabsorbent. The electrical parameter may be capacitance or resistance.

The sensor may comprise one or more colorimetric assay reagents.

Any of the systems may further comprising a wireless communicationdevice for transmitting wireless information corresponding to acharacteristic of the biofluid from the skin surface.

Any of the systems may have a flexible substrate that comprises amaterial selected from the group consisting of polydimethylsiloxane(PDMS), polyurethane, cellulose paper, cellulose sponge, polyurethanesponge, polyvinyl alcohol sponge, silicone sponge, polystyrene,polyimide, SU-8, wax, olefin copolymer, polymethyl methacrylate (PMMA),polycarbonate, polyvinyl chloride, chitosan, and any combinationthereof.

Any of the systems may further comprise an adhesive layer configured tomount the system to a skin surface, including an adhesive layer thatreversibly adheres the system to the skin surface. The adhesive layermay comprise medical grade acrylic or medical grade silicon.

Any of the systems may further comprise a protective layer embedded inor supported by the flexible substrate, such as a protective layer thatprevents biofluid from escaping from the reservoir chamber or the sweatsensor. The protective layer may comprise polyethylene.

Any of the collection layers may have an average thickness selected fromthe range of 50 μm to 1 mm. The collection layer may be a mesh. Thecollection layer may have a plurality of pores having an averagediameter selected from the range of 10 μm to 250 μm. The collectionlayer may comprise polyester.

Any of the systems may be incorporated into a glove.

Any of the systems by provide for a biofluid property that is visuallyobservable.

Any of the systems may provide a signal corresponding to the biofluidproperty is transmitted from the system to an external receiving device.

The biofluid property may be one or more of sweat volume, sweat rate, orsweat loss. The biofluid property may be pH. The biofluid property maycomprise the presence of, amount or concentration of an analyte in thebiofluid or component thereof.

The analyte may be an electrolyte, a metabolite, or a biomarker in thebiofluid or component thereof.

Any of the systems may provide a leading edge of biofluid in a sensormicrofluidic channel or reservoir that is sensed as a function of time.The leading edge may be sensed visually or measured using aphotodetector.

Any of the systems may further comprise an electronic sensor operablyconnected to the microfluidic network, wherein an amount of biofluid isproportional to an electrical resistivity or electrical conductivityparameter measured by the sensor.

Any of the systems may comprise a disposable portion comprising themicrofluidics network and a reusable portion corresponding to anelectronic device. wherein the disposable and reusable portions areconnected to each other by one or more selectively releasable couplingelements. The selectively releasable coupling elements may comprise amagnet.

Any of the systems may comprise a plurality of distinct component layersarranged in a stacked configuration.

Also provided herein are associated methods of using any of the systemsprovided herein, such as a method of analyzing biofluid from a subject,the method comprising the steps of: contacting the flexible substrate ofany systems provided herein with a skin surface of a subject; andanalyzing the biofluid from the skin surface. The contacting theflexible substrate is intended to be broad, and to include indirectcontact, such as by one or more intervening layers, such as adhesivelayers, capping layer, collecting layer, microfluidic layer. Contactingmay refer to conformal contact.

The biofluid may be sweat. The subject may be a human subject. The humansubject may be undergoing a diagnostic procedure or a therapeuticprocedure.

The subject may be a human subject monitoring the presence, onset orprogression of a disease condition or undergoing a fitness activity.

The method may further comprise the step of increasing biofluidretention in the system by one or more of: increasing biofluid viscosityin the microfluidic network; and/or absorbing biofluid to an absorbent.

The analyzing step may comprise: observing biofluid volume in at least aportion of the microfluidic network; and/or observing a colorimetricchange in a reservoir chamber.

The contacting step may comprise conformally contacting the flexiblesubstrate with the skin surface, and any intervening layers between theflexible substrate and the skin surface.

Without wishing to be bound by any particular theory, there may bediscussion herein of beliefs or understandings of underlying principlesrelating to the devices and methods disclosed herein. It is recognizedthat regardless of the ultimate correctness of any mechanisticexplanation or hypothesis, an embodiment of the invention cannonetheless be operative and useful.

Representative claims:

-   -   1. A microfluidic system for monitoring a biofluid property, the        system comprising:        -   a flexible substrate;        -   at least two microfluidic networks, each microfluidic            network configured to independently monitor a biofluid            property; wherein each microfluidic network comprises:            -   a microfluidic inlet conduit network at least partially                embedded in or supported by the substrate;            -   a biofluid inlet fluidically connected with the                microfluidic inlet conduit network to introduce a                biofluid from a skin surface to the microfluidic inlet                conduit during use;            -   a plurality of reservoir chambers, each reservoir                chamber fluidically connected with the microfluidic                inlet conduit network; and            -   a plurality of capillary burst valves fluidically                connected with the microfluidic conduit network, each                capillary burst valve positioned between fluidically                adjacent reservoir chambers.    -   2. The microfluidic system of claim 1, further comprising a        plurality of colorimetric sensors, wherein each colorimetric        sensor is positioned in a unique reservoir chamber to monitor        the biofluid property.    -   3. The microfluidic system of claim 1 or 2, wherein the at least        two microfluidic networks differ from each other by: (i) a        biofluid inlet dimension, (ii) a reservoir chamber volume of        each of the plurality of reservoir chambers, (iii) a burst        pressure of each of the plurality of capillary burst        valves, (iv) a chemical composition of a chemically-mediated        reaction chamber, or (v) any combination thereof.    -   4. The microfluidic system of any of claims 1-3, wherein a first        microfluidic network is configured to monitor a biofluid        parameter associated with a low-flow biofluid regime and a        second microfluidic network is configured to monitor a biofluid        parameter associated with a high-flow biofluid regime, and the        biofluid property is biofluid amount; biofluid analyte        concentration; biomarker presence or absence; or a combination        thereof.    -   5. The microfluidic system of claim 4, wherein the high biofluid        loss regime is at least 10 times greater than the low biofluid        loss regime.    -   6. The microfluidic system of any of claims 1-5, wherein each        microfluidic network further comprises: at least one        microfluidic outlet conduit, each microfluidic outlet conduit        fluidically connected to at least one of the plurality of        reservoir chambers and configured to relieve gas back pressure        from the microfluidic inlet conduit network.    -   7. The microfluidic system of any of claims 1-6, wherein the        plurality of reservoir chambers are chemically decoupled from        each other for independent biofluid property detection and/or        time sequential biofluid property monitoring.    -   8. The microfluidic system of any of claims 1-7, further        comprising a plurality of capillary burst valves, wherein at        least one capillary burst valve is positioned between        fluidically adjacent reservoir chambers.    -   9. A microfluidic system for measuring a biofluid pressure, the        system comprising:        -   a flexible substrate;        -   a plurality of reservoir networks at least partially            embedded in or supported by the flexible substrate, wherein            each reservoir network comprises:            -   a reservoir chamber;            -   a biofluid inlet fluidically connected to the reservoir                chamber via a capillary burst valve to introduce a                biofluid from a skin surface to the reservoir chamber,                wherein the capillary burst valve has a burst pressure;                and            -   an outlet fluidically connected to the reservoir                chamber;        -   wherein the burst pressure of each capillary burst valve is            selected to correspond to a pressure range of the biofluid            from the skin surface.    -   10. The system of claim 9, wherein at least one of the plurality        of reservoir networks has a unique capillary burst valve        pressure.    -   11. The system of any of claims 1-10, wherein the biofluid inlet        is fluidically aligned with a biofluid source of the skin        surface during use.    -   12. The system of any of claims 9-11, wherein each reservoir        network further comprises at least one colorimetric sensor to        provide an optical readout.    -   13. The system of any of claims 9-11, wherein at least a portion        of the capillary burst valves are fluidically aligned in a        serial configuration and have a burst valve pressure that        increases and spans a range that is greater than 0 kPa and less        than 10 kPa.    -   14. A microfluidic system to measure a biofluid property, the        system comprising:        -   a flexible substrate;        -   a microfluidic inlet conduit network at least partially            embedded in or supported by the flexible substrate;        -   a biofluid inlet fluidically connected to the microfluidic            inlet conduit network to introduce a biofluid from the skin            surface to the microfluidic inlet conduit during use;        -   a plurality of reservoir chambers, each reservoir chamber            fluidically connected with the microfluidic inlet conduit            network;        -   a plurality of capillary burst valves fluidically connected            with the microfluidic conduit network, each capillary burst            valve positioned between fluidically adjacent reservoir            chambers;        -   a microfluidic outlet conduit network fluidically connected            with the plurality of reservoir chambers and configured to            relieve gas back pressure from the microfluidic inlet            conduit network;        -   a plurality of colorimetric sensors, each positioned in a            unique reservoir chamber to measure a biofluid property; and            wherein: (i) at least one of the colorimetric sensors has a            color-responsive reagent to measure chloride.    -   15. The microfluidic system of claim 14, further comprising a        color indicator strip positioned between any two fluidically        adjacent reservoir chambers    -   16. The microfluidic system of claim 14, wherein: (i) at least        one of the colorimetric sensors has a color-responsive reagent        comprising silver chloranilate.    -   17. The microfluidic system of any of claims 14-16, wherein the        colorimetric sensors are configured to measure concentration of        chloride in the biofluid.    -   18. The microfluidic system of any of claims 1-17, further        comprising a capping layer connected to a skin-facing surface        and/or a back-facing surface of the flexible substrate.    -   19. A microfluidic system, comprising:        -   a flexible substrate having a skin-facing surface and a            back-facing surface;        -   a microfluidic network at least partially embedded in or            supported by the flexible substrate;        -   a sensor fluidically connected to the microfluidic network,            wherein the microfluidic network is configured to transport            a biofluid from a skin surface to the sensor; and        -   a capping layer, having a capping layer skin-facing surface            and a back-facing surface, wherein the back-facing surface            of the capping layer is attached to the skin-facing surface            of the substrate; wherein the flexible substrate is at least            partially formed of a thermoplastic elastomer or a polymer            configured to provide a high barrier to vapor or liquid            water transmission.    -   20. The microfluidic system of claim 18 or 19, wherein the        capping layer is at least partially formed of a thermoplastic        elastomer and an additive.    -   21. The microfluidic system of claim 20, wherein the flexible        substrate and the capping layer are formed of a common        thermoplastic elastomer composition.    -   22. The microfluidic system of any of claims 18-21, wherein the        flexible substrate and the capping layer have a common additive.    -   23. The microfluidic system of any of claims 19-22, wherein the        thermoplastic elastomer is a styrene copolymer selected from the        group consisting of styrene-ethylene-butadiene-styrene (SEBS),        styrene-isoprene-styrene (SIS), styrene-butadiene-styrene (SBS),        and any combination thereof.    -   24. The microfluidic system of any of claims 19-23, wherein the        thermoplastic elastomer has a weight fraction of styrene        copolymer selected from the range of 10% to 50%.    -   25. The microfluidic system of any of claims 19-24, wherein the        additive is a hydrocarbon compound characterized by a molecular        weight less than a user-selected molecular weight.    -   26. The microfluidic system of claim 25, wherein the additive is        paraffin oil.    -   27. The microfluidic system of any of claims 19-26, wherein the        thermoplastic elastomer has a weight ratio of additive to        styrene copolymer selected from the range of 1 to 3.    -   28. The microfluidic system of any of claims 18-27, wherein the        capping layer comprises a spatially distributed pattern of        relief, recess, or relief and recess features to achieve a        desired mechanical property while maintaining high barrier to        water vapor or liquid transmission.    -   29. The microfluidic system of claim 28, wherein the pattern        comprises a symmetrical pattern.    -   30. The microfluidic system of any of claim 28 or 29, wherein        the pattern is selected to achieve a desired mechanical property        of flexibility and stretchability of the capping layer that is        substantially matched to the flexible substrate.    -   31. The microfluidic system of claim 30, wherein the mechanical        property is Young's modulus of less than 100 MPa, a net bending        stiffness of less than 1 nN m, and/or a thickness of less than 5        mm.    -   32. The microfluidic system of any of claims 28-31, wherein the        pattern is spatially aligned with at least a portion of the        microfluidic network.    -   33. The microfluidic system of any of claims 18-32, wherein the        capping layer is at least partially formed of a rigid polymer        selected from the group consisting of a polyolefin, a polyester,        a fluorocarbon, a polyamide, a polyimide, and any combination        thereof.    -   34. The microfluidic system of claim 33, wherein: the polyolefin        is selected from the group consisting of polyethylene,        polypropylene and polyisobutylene; the polyester is selected        from the group consisting of polyethylene terephthalate and        polyethylene naphthalate; the fluorocarbon is selected from the        group consisting of polyvinylidene chloride and        polytetrafluoroethylene; the polyamide is a nylon; and/or the        polyimide is a poly-oxydiphenylene-pyromellitimide.    -   35. The microfluidic system of any of claims 18-34, further        comprising an adhesive layer on the skin facing surface of the        capping layer; wherein the adhesive layer comprises an adhesive        compound capable of reversibly adhering the system to the skin        surface.    -   36. The microfluidic system of claim 35, wherein the adhesive        layer comprises medical-grade acrylic.    -   37. The microfluidic system of any of claims 35-36, wherein the        substrate, the capping layer, the adhesive compound, or any        combination thereof further comprise a tackifier additive.    -   38. The microfluidic system of claim 37, wherein the substrate,        the capping layer, or both the functional substrate and the        capping layer have a weight fraction of tackifier additive of        between 30% to 80%.    -   39. The microfluidic system of any of claims 37-38, wherein the        tackifier additive is rosin gum.    -   40. The microfluidic system of any of claims 19-39, wherein the        microfluidic network comprises a plurality of reservoirs and a        microfluidic inlet conduit network having a biofluid inlet to        introduce the biofluid to the microfluidic network; and wherein        the microfluidic outlet conduit network is fluidically connected        to the plurality of reservoirs.    -   41. The microfluidic system of claim 40, wherein the        microfluidic network further comprises a microfluidic outlet        conduit network fluidically connected to the plurality of        reservoirs, the microfluidic inlet conduit network, and an        outlet, and wherein the outlet is configured to (i) provide for        release of gas back pressure from the microfluidic inlet conduit        network, and (ii) prevent ingress of a liquid from a surrounding        environment into the microfluidic outlet conduit network.    -   42. The microfluidic system of any of claim 40 or 41, wherein        the sensor is a colorimetric sensor.    -   43. The microfluidic system of any of claim 40 or 41, wherein        the sensor is an electrochemical sensor.    -   44. The microfluidic system of any of claims 19-43 comprising        two or more sensors, including at least one colorimetric sensor        and one electrochemical sensor.    -   45. The microfluidic system of claim 44, wherein the        colorimetric sensor is positioned in one of the plurality of        reservoirs.    -   46. The microfluidic system of claim 44 or 45, wherein the        electrochemical sensor is positioned in one of the plurality of        reservoirs.    -   47. The microfluidic system of any of claims 1-46, further        comprising a biofluid gelling additive or an absorbent contained        within the microfluidic network.    -   48. The microfluidic system of claim 47, wherein the biofluid        gelling additive comprises two or more unique biofluid gelling        additives.    -   49. The microfluidic system of claim 47 or 48, wherein the        biofluid gelling agent is configured to mix or react with the        biofluid to increase a biofluid viscosity.    -   50. The microfluidic system of claim 49, wherein, the increase        in biofluid viscosity is by at least a factor of 2 of the        biofluid viscosity before mixing or reacting with the biofluid        gelling agent.    -   51. The microfluidic system of any of claims 47-50, wherein the        biofluid gelling agent comprises cellulose or a derivative        thereof.    -   52. The microfluidic system of claim 51, wherein the biofluid        gelling agent is methyl cellulose or hydroxypropyl        methylcellulose.    -   53. The microfluidic system of any of claims 47-52, wherein the        weight ratio of the biofluid gelling agent to biofluid, in at        least one of the plurality of reservoirs, is selected from the        range of 0.1 to 1.    -   54. The microfluidic system of any of claims 1-53, wherein the        substrate is a functional substrate.    -   55. A microfluidic system, comprising:        -   a flexible substrate;        -   a microfluidic network at least partially embedded in or            supported by the flexible substrate;        -   a sensor fluidically connected to the microfluidic network,            wherein the microfluidic network is configured transport a            biofluid from a skin surface to the sensor; and        -   a biofluid gelling additive or a biofluid absorbent            contained in the microfluidic network to reduce biofluid            loss from the microfluidic network.    -   56. The microfluidic system of claim 55, wherein the        microfluidic network comprises:        -   a plurality of reservoirs;        -   a biofluid inlet to introduce a biofluid to the microfluidic            network; and        -   a microfluidic inlet conduit network fluidically connected            to the biofluid inlet and the plurality of reservoirs to            introduce a biofluid to the reservoirs.    -   57. The microfluidic system of claim 56, wherein the        microfluidic network further comprises:        -   a microfluidic outlet conduit network fluidically connected            to the plurality of reservoirs; and        -   an outlet fluidically connected to the microfluidic outlet            conduit;        -   wherein the outlet is configured to:            -   provide for release of gas back pressure from the                microfluidic inlet conduit network, and            -   prevent ingress of a liquid from an environment                surrounding the system into the microfluidic outlet                conduit network.    -   58. The microfluidic system of any of claims 55-57, wherein the        sensor is a colorimetric sensor.    -   59. The microfluidic system of any of claims 55-57, wherein the        sensor is an electrochemical sensor.    -   60. The microfluidic system of any of claims 55-59, comprising        two or more sensors.    -   61. The microfluidic system of claim 58, wherein the        colorimetric sensor is positioned in one of the plurality of        reservoirs.    -   62. The microfluidic system of claim 59, wherein the        electrochemical sensor is positioned in one of the plurality of        reservoirs.    -   63. The microfluidic system of any of claims 55-62, wherein the        biofluid gelling additive is positioned in at least one of the        plurality of reservoirs.    -   64. The microfluidic system of any of claims 55-63, comprising        two or more biofluid gelling additives.    -   65. The microfluidic system of any of claims 55-64, wherein the        biofluid gelling agent is configured to mix or react with the        biofluid to increase a biofluid viscosity.    -   66. The microfluidic system of claim 65, wherein, the increase        in biofluid viscosity is by at least a factor of 2 of the        biofluid viscosity before mixing or reacting with the biofluid        gelling agent.    -   67. The microfluidic system of any of claims 55-66, wherein the        biofluid gelling agent is at least partially formed of cellulose        or a derivative thereof.    -   68. The microfluidic system of claim 67, wherein the biofluid        gelling agent is methyl cellulose or hydroxypropyl        methylcellulose.    -   69. The microfluidic system of any of claims 55-68, wherein the        weight ratio of the biofluid gelling agent to biofluid, in at        least one of the plurality of reservoirs, is selected from the        range of 0.1 to 1.    -   70. The microfluidic system of any of claims 55-69, further        comprising a capping layer, having a capping layer skin facing        surface and a back surface, wherein the back surface is affixed        to a skin facing surface of the substrate.    -   71. The microfluidic system of any of claims 55-70, wherein the        substrate is at least partially formed of a thermoplastic        elastomer having an additive.    -   72. The microfluidic system of any of claims 70-71, wherein the        capping layer is at least partially formed of a thermoplastic        elastomer and an additive.    -   73. The microfluidic system of claim 72, wherein the substrate        and the capping layer each comprise: a common thermoplastic        elastomer composition, or a different thermoplastic elastomer        composition.    -   74. The microfluidic system of any of claims 72-73, wherein the        substrate and the capping layer have a common additive.    -   75. The microfluidic system of any of claims 71-74 wherein the        thermoplastic elastomer is a styrene copolymer selected from the        group consisting of styrene-ethylene-butadiene-styrene (SEBS),        styrene-isoprene-styrene (SIS), styrene-butadiene-styrene (SBS),        and any combination thereof.    -   76. The microfluidic system of claim 75, wherein the        thermoplastic elastomer has a weight fraction of styrene        copolymer selected from the range of 10% to 50%    -   77. The microfluidic system of any of claims 71-76, wherein the        additive is a hydrocarbon compound characterized by a molecular        weight less than a user-selected molecular weight.    -   78. The microfluidic system of claim 77, wherein the additive is        paraffin oil.    -   79. The microfluidic system of any of claims 70-78, wherein the        thermoplastic elastomer has a weight ratio of additive to        styrene copolymer selected from the range of 1 to 3.    -   80. The microfluidic system of any of claims 70-79, wherein the        capping layer comprises a spatially distributed pattern of        relief, recess, or relief and recess features to achieve a        desired mechanical property while maintaining high barrier to        water vapor or liquid transmission.    -   81. The microfluidic system of claim 80, wherein the pattern        comprises a symmetrical pattern.    -   82. The microfluidic system of any of claim 80 or 81, wherein        the pattern is selected to achieve a desired mechanical property        of flexibility and stretchability of the capping layer that is        substantially matched to the flexible substrate, wherein the        mechanical property is one or more of a Young's modulus, a        bending stiffness or an average thickness.    -   83. The microfluidic system of any of claims 81-82, wherein the        pattern is spatially aligned with at least a portion of the        microfluidic network.    -   84. The microfluidic system of any of claims 55-83 wherein the        capping layer is at least partially formed of a rigid polymer        selected from the group of a polyolefin, a polyester, a        fluorocarbon, a polyamide, a polyimide, and any combination        thereof.    -   85. The microfluidic system of claim 84, wherein: the polyolefin        is selected from the group consisting of polyethylene,        polypropylene and polyisobutylene; the polyester is selected        from the group consisting of polyethylene terephthalate and        polyethylene naphthalate; the fluorocarbon is selected from the        group consisting of polyvinylidene chloride and        polytetrafluoroethylene; the polyamide is a nylon; and/or the        polyimide is a poly-oxydiphenylene-pyromellitimide.    -   86. The microfluidic system of any of claims 55-85, further        comprising an adhesive layer on the skin facing surface of the        capping layer; wherein the adhesive layer comprises an adhesive        compound capable of reversibly adhering the system to the skin        surface.    -   87. The microfluidic system of claim 86, wherein the adhesive        layer comprises medical-grade acrylic.    -   88. The microfluidic system of any of claims 86-87, wherein the        substrate, the capping layer, the adhesive layer, or any        combination thereof further comprise a tackifier additive.    -   89. The microfluidic system of claim 88, wherein the substrate,        the capping layer, or both the substrate and the capping layer        have a weight fraction of tackifier additive of between 30% to        80%.    -   90. The microfluidic system of any of claims 88-89, wherein the        tackifier additive is rosin gum.    -   91. The microfluidic system of any of claims 1-80, further        comprising an expunge port fluidically connected with the        reservoir chamber for the removal of biofluid from the reservoir        chamber.    -   92. An epidermally-mountable microfluidic system for measuring a        characteristic of a biofluid from a skin surface; the system        comprising:        -   a flexible substrate;        -   a biofluid inlet embedded on or supported by the substrate            for receiving the biofluid from the skin surface; and        -   a microfluidic channel fluidically connected to the biofluid            inlet for receiving at least a portion of a biofluid from            the biofluid inlet, the microfluidic channel having a            patterned grating;        -   wherein transmission of incident electromagnetic radiation            through the patterned grating changes as a function of a            biofluid amount in the microfluidic channel.    -   93. The system of claim 92, wherein the epidermal microfluidic        system further comprises an indicator in optical communication        the patterned grating; wherein changes in the transmission of        incident electromagnetic radiation through the grating changes        the appearance of the indicator.    -   94. The system of any of claims 92-93, wherein the patterned        grating comprises a hydrophilic polymer; and wherein        transmission of the incident electromagnetic radiation by the        patterned grating increases when the chamber is filled with the        biofluid.    -   95. The system of any of claims 92-93, wherein the patterned        grating comprises a hydrophobic polymer; and wherein        transmission of the incident electromagnetic radiation by the        patterned grating decreases when the chamber is filled with the        biofluid.    -   96. The system of any of claims 92-95, wherein the epidermal        microfluidic system further comprises an expunge port        fluidically connected with the reservoir chamber for the removal        of the biofluid from the reservoir chamber.    -   97. The system of any of claims 92-96, further comprising an        adhesive layer.    -   98. The system of claim 97, wherein the adhesive layer comprises        an adhesive capable of reversibly adhering to the skin surface.    -   99. The system of claim 97, wherein the adhesive layer comprises        medical grade acrylic or medical grade silicone.    -   100. The system of any of claims 96-99, wherein the expunge port        comprises two outlets.    -   101. The system of any of claims 96-100, further comprising a        capillary burst valve fluidically connected to said expunge port        and said reservoir chamber.    -   102. The system of claim 101, wherein said capillary burst valve        is positioned between said expunge port and said reservoir        chamber.    -   103. The system of any of claims 92-102, wherein said patterned        grating is nanopatterned or micropatterned.    -   104. An epidermal microfluidic system for measuring a        characteristic of a biofluid; the system comprising:        -   a flexible substrate;        -   a collection layer embedded in or supported by the flexible            substrate, wherein the collection layer promotes transport            of the biofluid from the skin surface;        -   at least one reservoir chamber embedded in or supported by            the flexible substrate and fluidically connected to the            collection layer, the reservoir chamber having:            -   an absorbent provided to receive at least a portion of                the biofluid from the collection layer; and            -   a sensor for measuring a characteristic of the biofluid                received by the absorbent; wherein the absorbent                provides a force for transporting the biofluid that is                greater than a capillary force of the collection layer                for transporting the biofluid.    -   105. An epidermal microfluidic system for measuring a        characteristic of a biofluid, the system comprising:        -   a flexible substrate;        -   a radiofrequency (RF) heater embedded in or supported by the            flexible substrate; wherein the RF heater is capable of            increasing a temperature of the skin surface, thereby            increasing the release rate of the biofluid;        -   at least one sensor embedded in or supported by the flexible            substrate to measure the characteristic of the biofluid.    -   106. The epidermal microfluidic system of claims 104-105,        wherein the biofluid characteristic is amount of sweat loss or        presence or absence of a biomarker from a skin surface.    -   107. The system of any of claims 104-106, wherein the sensor is        an electronic sensor.    -   108. The system of claim 107, wherein the electronic sensor        comprises one or more high sensitivity electrodes configured to        measure a change in an electrical parameter caused by biofluid        received by the absorbent.    -   109. The system of claim 108, wherein the electrical parameter        is capacitance.    -   110. The system of any of claims 104-109, wherein the sensor        comprises one or more colorimetric assay reagents.    -   111. The system of any of claims 104-110, further comprising a        wireless communication device for transmitting wireless        information corresponding to a characteristic of the biofluid        from the skin surface.    -   112. The system of any of claims 1-111, wherein said flexible        substrate comprises a material selected from the group        consisting of polydimethylsiloxane (PDMS), polyurethane,        cellulose paper, cellulose sponge, polyurethane sponge,        polyvinyl alcohol sponge, silicone sponge, polystyrene,        polyimide, SU-8, wax, olefin copolymer, polymethyl methacrylate        (PMMA), polycarbonate, polyvinyl chloride, chitosan, and any        combination thereof.    -   113. The system of any of claims 1-112, further comprising an        adhesive layer configured to mount the system to a skin surface.    -   114. The system of claim 113, wherein the adhesive layer        reversibly adheres the system to the skin surface.    -   115. The system or claim 114, wherein the adhesive layer        comprises medical grade acrylic or medical grade silicon.    -   116. The system of any of claims 1-115, further comprising a        protective layer embedded in or supported by the flexible        substrate.    -   117. The system of claim 116, wherein the protective layer        prevents biofluid from escaping from the reservoir chamber or        the sweat sensor.    -   118. The system of claim 116, wherein said protective layer is        polyethylene.    -   119. The system of claim 104, wherein the collection layer has        an average thickness selected from the range of 50 μm to 1 mm.    -   120. The system of claim 104 or 119, wherein the collection        layer is a mesh.    -   121. The system of claim 104 or 119, wherein the collection        layer has a plurality of pores having an average diameter        selected from the range of 10 μm to 250 μm.    -   122. The system of claim 104 or 119, wherein the collection        layer is polyester.    -   123. The system any of claims 1-122 that is incorporated into a        glove.    -   124. The system of any of claims 1-123, wherein the biofluid        property is visually observable.    -   125. The system of any of claims 1-123, wherein a signal        corresponding to the biofluid property is transmitted from said        system to an external receiving device.    -   126. The system of any of claims 1-125, wherein the biofluid        property is one or more of sweat volume, sweat rate, or sweat        loss.    -   127. The system of any of claims 1-125, wherein the biofluid        property is pH.    -   128. The system of any of claims 1-125, wherein the biofluid        property comprises the presence of, amount or concentration of        an analyte in said biofluid or component thereof.    -   129. The system of claim 128, wherein said analyte is an        electrolyte, a metabolite, or a biomarker in said biofluid or        component thereof.    -   130. The system of any of claims 1-129, wherein a leading edge        of biofluid in a sensor microfluidic channel or reservoir is        sensed as a function of time.    -   131. The system of claim 130, wherein the leading edge is sensed        visually or measured using a photodetector.    -   132. The system of any of claims 1-131, wherein the flexible        substrate is a functional substrate.    -   133. The system of any of claims 1-132, further comprising an        electronic sensor operably connected to the microfluidic        network, wherein an amount of biofluid is proportional to an        electrical resistivity or electrical conductivity parameter        measured by the sensor.    -   134. The system of any of claims 1-133, comprising a disposable        portion comprising the microfluidics network and a reusable        portion corresponding to an electronic device. wherein the        disposable and reusable portions are connected to each other by        one or more selectively releasable coupling elements.    -   135. The system of claim 134, wherein the selectively releasable        coupling elements comprise a magnet.    -   136. The system of any of claims 1-135, comprising a plurality        of distinct component layers arranged in a stacked        configuration.    -   137. A method of analyzing biofluid from a subject, the method        comprising the steps of:        -   contacting the flexible substrate of any claims 1-136 with a            skin surface of a subject; and        -   analyzing the biofluid from the skin surface.    -   138. The method of claim 137, wherein said biofluid is sweat.    -   139. The method of any of claims 137-138, wherein said subject        is a human subject.    -   140. The method of any of claims 137-138, wherein said subject        is a human subject undergoing a diagnostic procedure.    -   141. The method of any of claims 137-138, wherein said subject        is a human subject undergoing a therapeutic procedure.    -   142. The method of any of claims 137-138, wherein said subject        is a human subject monitoring the presence, onset or progression        of a disease condition.    -   143. The method of any of claims 137-138, wherein said subject        is a human subject undergoing a fitness activity.    -   144. The method of any of claims 137-138, further comprising the        step of increasing biofluid retention in the system by one or        more of:        -   increasing biofluid viscosity in the microfluidic network;            and/or        -   absorbing biofluid to an absorbent.    -   145. The method of any of claims 137-144, wherein the analyzing        comprises: observing biofluid volume in at least a portion of        the microfluidic network; and/or observing a colorimetric change        in a reservoir chamber.    -   146. The method of any of claims 137-145, wherein the contacting        comprises conformally contacting the flexible substrate with the        skin surface, and any intervening layers between the flexible        substrate and the skin surface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows: (a) Schematic illustrations and optical images (inset) ofthin, soft microfluidic devices for chrono-sampling of sweat. (b)Exploded view illustration of a device and its interface with skin. (c)Top view illustration of microfluidic channels filled with blue-dyedwater. (d) Optical images of in vitro testing of chrono-sampling ofblue-dyed water in a working device.

FIG. 2 shows: (a) Detailed schematic illustration of a unit cell in adevice, including a collection chamber, extraction chamber, samplingoutlet, and three capillary bursting valves (CBV) and SEM images ofCBVs. (b) Sketch of capillary bursting valves with indicated channelwidth and diverging angle. (c) Measurement of contact angle of water onPDMS as a function of aging time after oxygen plasma treatment. (d)Experimental results (bars) and theoretical values (square box) ofcapillary bursting pressure of CBV #1, #2, and #3 of device after 1 dayfrom oxygen plasma treatment. (e) Optical images and schematicillustrations of the working principle of the capillary bursting valvesfor chrono-sampling. (i) Before entering collection chamber (ii) fillingthe collection chamber (iii) flowing to next chamber (iv) aftercentrifugation.

FIG. 3 shows: a process of recovering liquid from chrono-samplingdevice.

FIG. 4 shows microfluidic systems illustrating a variety of exampledesigns of the systems disclosed herein, including, for example,different amounts and shapes of the reservoir chambers.

FIG. 5 shows two example microfluidic system having two microfluidicnetworks. Differences between two microfluidic networks in a singlemicrofluidic system may include capillary burst valve burst pressure(e.g., size), reservoir chamber size and shape, and inlet size, therebyproviding a high and low loss biofluid regime in a single system.

FIG. 6 shows: A. Schematic to measure pressure of sweat from sweat glandusing capillary bursting valve; B The exploded view of the device havinga capping layer, microfluidic channel with CBVs and adhesive layer; CFabrication steps for the device.

FIG. 7 shows: A schematic of the pressure measurement device with 12different CBV; B The scanning electron micrograph (SEM) images of theCBVs, showing different sizes of different CBVs; C the opticalmicroscopic images of blue dyed filled CBVs. D optical image of bluedyed filled device and CoCl₂ applied device on the skin.

FIG. 8 shows: (a) the water contact angle on the PDMS; (b) The criticaladvancing contact angle of water on the PDMS; (c) the SEM images ofrounding edge of the CBVs; (d) the bursting pressure of the CBVs from invitro test and numerical calculation.

FIG. 9 shows: (a) the optical images of measurement of pressure of sweatgland from exercising and thermal exposure in sauna; (b) the graph ofthe measured pressure of the sweat gland with different condition andregion of the body.

FIG. 10 shows colorimetric chloride concentration detection using silverchloranilate in a microfluidic system.

FIG. 11 shows: (a) color development in the chamber with silverchloranilate according to the chloride concentrations; (b) graph of theextracted color value from the chamber in (a); (c) schematic of colorcalibration marker.

FIG. 12 shows detection of chloride concentration in the biofluid usingthe color marker (indicator) strip: (a) color level from the chambersand color calibration marker; (b) measuring the chloride concentrationusing color level.

FIG. 13 shows images and tables corresponding to in vitro and in situtests with a microfluidic system, illustrating the accuracy of chloridedetection using silver chloranilate.

FIG. 14. a) Detailed schematic illustration of a unit cell in a device,including a collection chamber, sampling outlet, and four capillarybursting valves (CBV). b) Sketch of capillary bursting valves withindicated channel width and diverging angle. c) Experimental results(bars) and theoretical values (asterisk) of capillary bursting pressureof CBV #1, #2, #3 and #4 of device. d) Optical images of the workingprinciple of the capillary bursting valves for chrono-sampling. (i)Before entering collection chamber (ii) filling the collection chamber#1 (iii) flowing to chamber #2 (iv) flowing to next chamber (v) afterfilling all chambers. e) Optical images of in vitro testing ofchrono-sampling with different colored dyes in water (red, green, andblue) and enlarged images of the interface between different coloreddye.

FIG. 15 illustrates cross sections of certain embodiments of themicrofluidic systems disclosed herein. The data plot shows fluid lossdue to evaporation from the microfluidic systems as a function of time.The data shows that water may evaporate through PDMS and that a cappinglayer, including a PET capping layer for example, reduces fluid loss.

FIG. 16. Comparison of evaporation rate of sweat after collection. A)Experimental setup for measuring evaporative water loss from epifluidicdevices. B) Cross-sectional micrograph of an SIS device. C)Cross-sectional micrograph of a PDMS device. D) The mass change of PDMSand SIS epifluidic devices after filling with water and heating at 37°C. SIS devices are able to store sweat for 4 hours with less than 20%loss, while PDMS devices lost ˜100% within 3 hrs.

FIG. 17 Left: Cross section of a single microfluidic chamber containinga viscosity modifier (HPMC). Right. Comparison of a chamber filled withsweat with and without HPMC. Pressing the chamber without the modifiercauses the CBV's to burst releasing the captured sweat. The chamber withHPMC does not burst in response to pressing, reflecting additionalstability to the chamber and burst valve with a viscosity modifier.

FIG. 18. The soft mechanics, electronics, and hydrophilic microporousgeometries of the devices enable them to reliably fit to the skin and tocapture and detect the nanoliter volume of insensible sweat releasedfrom skin efficiency (Panel a) during resting. A representative deviceshown in Panel b has a rectangular geometry (the height, width andlength are 930 μm, 2 cm and 4 cm) and two detection areas: one comprisesan electronic system for detecting and measuring the amount ofinsensible sweat loss with time, and the other comprises absorbents(e.g., microporous polymers) for capturing and collecting insensiblesweat from skin to analyze biomarkers. The device can be attached to anypart of body.

FIG. 19. A representative sweat inducing system with RF heaters forcollecting sweat efficiency, with the system incorporated into a glove.

FIG. 20. (A) Schematic illustrating the exploded view of a hybridbattery-free system. Close-up image of (B) micro-fluidic patch withembedded sensors (C) battery-free NFC electronics. (D) Imageillustrating the reversible magnetic attachment of the NFC electronicsto the micro-fluidic patch. (E) Image of the complete system (F) Imageillustrating device during sweating. (G) A phone interface thatillustrates wireless communication and image acquisition.

FIG. 21. Electrical characterization of NFC electronics. (A) Simplifiedschematic of electrochemical sensor readout. (B) Image illustrating thedevice bent at decreasing radii. (C) I-V measurements of shorted sensorsrecoded with decreasing curvature radii (D) Phase response measurementsof NFC electronics with decreasing radii. (E) I-V measurements ofshorted sensors with repeated attachment and detachment of theelectronics to the micro-fluidics. (F) Impedance of magnetic contactsover a wide range of frequencies. Effect of (G) distance and (H) anglebetween NFC reader and device on signal recording.

FIG. 22. Lactate sensor characterization. (A) Exploded-view schematicvisualizing layer makeup of the fuel cell-based lactate sensor. (B)Image of the actual lactate sensor. (C) Real-time sensor response toincreasing lactate concentration in phosphate buffer (pH 7.0) at 25° C.and (D) the corresponding calibration (n=3). (E) Plot illustratingreversible sensor response for lactate sensor for four consecutivecycles of varying lactate concentration. (Inset: Calibration plotcomparing the sensor signal plotted in (E) for the four cycles. V:voltage in millivolts; C: concentration in millimolar). (F) Real-timedata acquired for increasing lactate concentration in artificial sweatunder common physiological sweat conditions (temperature=30° C.;pH=5.5). (G) Calibration plot obtained for lactate sensors in artificialsweat at different pH (n=3).

FIG. 23. Glucose sensor characterization. (A) Exploded-view schematicvisualizing layer makeup of the fuel cell-based glucose sensor. (B)Image of the actual glucose sensor. (C) Real-time sensor response toincreasing glucose concentration in phosphate buffer (pH 7.0) at 25° C.and (D) the corresponding calibration. (n=3) (E) Plot illustratingreversible sensor response for glucose sensor for four consecutivecycles of varying glucose concentration. (Inset: Calibration plotcomparing the sensor signal plotted in (E) for the four cycles. V:voltage in millivolts; C: concentration in micromolar). (F) Real-timedata acquired for increasing glucose concentration in artificial sweatunder common physiological sweat conditions (temperature=30° C.;pH=5.5). (G) Calibration plot obtained for glucose sensors in artificialsweat at different pH (n=3).

FIG. 24. Colorimetric assay characterization. Calibration andcorresponding color evolution for physiologically relevant levels of (A)chloride (n=3) and (B) pH (n=3). (C) Filling of sweat rate sensor. (D)Image illustrating chrono-sampling feature of the micro-fluidics system.

FIG. 25. (A and B) SPICE schematic of amplification scheme where R3 andR2 represent contact resistance of magnetic connection and R1 representsthe respective load for (A) lactate and (B) glucose biofuel cell-basedsensor. (C-D) Simulation results for amplified signal (black trace forvoltage and blue trace for sensor current) vs. benchtop measurements(red trace) with oscillating supply voltage; demonstrating supplyvoltage insensitivity for (C) lactate and (D) glucose measurements withincreasing concentrations.

FIG. 26. Color reference marker of a biofluid property that is chlorideconcentration (mM) and pH.

FIG. 27. Relationship between normalized total sweat loss and volumecaptured in sweat rate sensor.

FIG. 28. (A) Schematic illustration of capillary bursting valves in acolorimetric detection chamber. (B) Schematic illustration ofmicrofluidic channel: 1) Glucose detection chamber 2) lactate detectionchamber 3) chloride chrono detection chambers 4) pH chrono detectionchambers 5) sweat rate detection chamber. In this manner, any of thesystems may be characterized as providing multiplexed detection,including with respect to time.

FIG. 29 illustrates a nano/micro patterned grating in a hydrophilicpolymer which diffuses light when the chamber is empty, but transmitslight when the chamber is full. A micropatterned grating in ahydrophilic surface scatters light when there is a refractive indexmismatch and transmits light when the mismatch is negligible.

FIG. 30 illustrates a similar concept, but utilizes a hydrophobicpolymer surface and a nano/micro patterned grating to trap air bubbleswhich reflect light when the chamber is full. Patterned features in ahydrophobic surface trap air bubbles when filled with sweat and reflectincident light, changing the appearance of the colored indicator.

FIG. 31 illustrates a means of resetting the sensor to its initialstate. Dual expunge ports reduce the likelihood of accidental sweatdischarge, but when covered and pressed simultaneously empty the chamberand reset the device back to its initial state.

FIG. 32. Purge system for resettable sweat device. A) Cross sectionschematic showing the manually-actuated valves. B) Step 1: Sweat isextracted from the channel via pinching and opening valve 1 bystretching the top elastomeric membrane. This creates suction whichextracts sweat from the chamber. C) Step 2: Seal valve 1 by pressing. D)Open valve 2 and purge sweat. E) The device is ready to be reused.

FIG. 33. Sweat induction through hot shower/bath (e.g., extrinsicheater). (a) Instructions and procedures of how to induce and collectsweat through hot shower/bath. (b) (I-IV) Schematic illustration of thepositions of the devices and a demonstration of sweat collection resultin human study of one subject. Diameter of sweat harvesting area: redcircle: 5 mm, blue circle: 3 mm. (c) Number of filled reservoirs frominlet 1-3 on forehead, chest, axilla, and back. Black dash lineindicates the minimum volume/reservoir needed for colorimetric analysis.

FIG. 34 illustrates microfluidic networks optimized to be filled and tostore biofluids in discrete amounts without the trapping of air to fullyextract the biofluid with approximately 100% efficiency.

FIG. 35 illustrates the benefit of the design combining a pipe into abowl shape for facile fluid filling or extraction without air entrapmentwhile providing clear visual volume information.

FIG. 36 shows an exemplary multilayer device having separate layercomponents in a stacked configuration. The layers can be individuallypatterned to facilitate any number of desired functionality, such asfluid transport through an adhesive layer, collection and analysis(analytics layer), fluid barrier layer (capping layer) with an outletport for removal of biofluid.

FIG. 37. Top panel (labeled as A) is a schematic of a collection device.(B) The device collection layer (1) comprises three independent chambers(e.g., three individual microfluidic networks), each with an inlet andoutlet.

FIG. 38. Optical image of the device, including undergoing bending andtwisting, panels A-C, respectively, also referred herein as beingflexible.

FIG. 39. Optical image of the device filling with sweat (teal) over a 30min collection period after pilocarpine iontophoresis.

FIG. 40. Optical image sequence showing extraction of sweat from afilled device A. Once removed from the skin, a standard pipette providessufficient negative pressure to extract sweat B. Device geometryoptimized to fully extract sweat C.

FIG. 41. Schematic of a multilayer device in a stack configuration where(A) is a capping layer, (B) is a colorimetric analysis layer, (C) is acollection layer, (D) is a skin interface layer. (B) and (C) areindependent of each other providing on-board analysis of sweat collectedfor external analysis.

FIG. 42. Representative device fabricated using a multistackconstruction.

FIG. 43. Resettable sweat indicator via light scattering. A) Scatteringmaterial patterned in the channel of a microfluidic device causes lightscattering and presents a white color. B) Captured sweat enters themicrochannel and reduces scattering and presents the color of theindicator (black). The scattering media has a comparable refractiveindex to sweat. C) Extracting sweat resets the device to the initialstate.

FIG. 44. a) Optical images of soft and flexible microfluidic devices forcolorimetric analysis of sweat on the skin (top) and under mechanicaldiction of bending (bottom left) and twisting (bottom right). b) Topview illustration of microfluidic channels filled with blue-dyed water.c) Exploded view illustration of a device and its interface with skin.d) Procedure of collecting sweat sample and color analysis of digitalimage of the device.

FIG. 45. a) Schematic illustration of device with color referencemarkers of chloride, glucose, pH and lactate, and number for indicatingsweat collection volume. b) Optical images color development ofthermochromic liquid crystal temperature sensor according to temperature(top) and color level of each color (bottom). Optical images colordevelopment of assay chambers according to sample concentrations (top)and color level of each color (bottom) of c) chloride, d) glucose, e) pHand f) lactate.

FIG. 46. The structure of temperature sensing film of thermochromicliquid crystal.

FIG. 47. Color development at various flow rate.

FIG. 48. Color development at different channel depth.

FIG. 49. a) Schematic illustration of in vitro accuracy test of colorreference marker in various light sources of white light bulb, yellowlight bulb, and daylight. Measured concentration using color referencemarker in the device filled with standard solutions of b) chloride, c)glucose, d) pH, and e) lactate marker in various light sources.

FIG. 50. Schematic illustration and digital images of the microfluidicdevice for sweat chloride, sodium, and zinc sensing by fluorometricmethods. a) Schematic illustrating the exploded view of themicro-fluidic device for fluorescence assays. b) Image illustratingfluorescence signals of chloride, sodium, and zinc probes on the deviceunder the excitation light. Image illustrating c) the peeling of thedetachable black shield from the microfluidic device and d) themechanical flexibility under mechanical distortions: forward twisting(left) and backward twisting (center), and on the palm (right).

FIG. 51. Description of the design of the micro-fluidic channel. (a)Detailed schematic illustration of a unit cell in a sweat device with areservoir and three capillary bursting valves. (b) The Young-Laplaceequation for calculating the bursting pressures (BP) of the valves. (c)Calculated BP of the three valves and the required parameters for thecalculation.

FIG. 52. Description of the design of the smartphone based fluorometricimaging system. (a) Image illustrating the overall concept offluorescence-imaging system with a smartphone-attached accessory. (b)Image of the photographed of smartphone attachment with the dark box andexcitation/emission filters. (c) Image illustrating the fluorometricimaging system in the interfaces of smartphone and the filters. Spectraof smartphone LED light with excitation filters (two dark bluetransparent filters) (d) and without filter (e).

FIG. 53. (a) Procedure of the fluorometric assay: 1. Collection of sweatusing a sweat device 2. Peeling the black shield 3. Taking a photo ofthe device using a smartphone attached accessory. (b) Method offluorescence calibration. Fluorescence reference material consisting ofan ionic liquid and a green fluorescence dye (c) and red reference dye(d).

FIG. 54. Effect of white PDMS on fluorescence intensity. (a) Differenceof the fluorescence image between white and transparent PDMS devices.(b) Schematic illustrating the reflection of fluorescence by thetitanium oxide particles included in the white PDMS. (c) SEM image of awhite PDMS.

FIG. 55. Fluorescence images of chloride, zinc, and sodium assays andits light intensity dependence on the concentration. (a) Imageillustrating the micro reservoirs for the assays before (upper) andafter (lower) filled up with sweat under visible light. Changes of thefluorescence and its normalized intensity at various concentrations of(b) chloride, (c) sodium, and (d) zinc.

FIG. 56. (a) Photograph of a subject wearing a micro-fluidic patchduring sweat testing. Images of the sweat patch without the black shieldafter sweat collection under (b) visible light and (c) the blue lightemitted by a smartphone. (d) Calculated concentrations of sweat (d)chloride (green closed circles), (e) sodium (blue closed circles), and(f) zinc (pink closed circles) with the estimated sweat loss (blackdotted lines). solid green, blue and pink lines indicate theconcentrations measured by ion chromatography for chloride, ICP-MS forzinc, and atomic absorption spectrometry for sodium in the sweat. (g)Changes of estimated sweat loss with being filled up the microreservoirs and center microchannel structures.

FIG. 57. Fluorometric chloride assay using 0.3 μL of artificial sweatcontaining 0-150 mM chloride.

FIG. 58. (a)-(b) Optical images of soft and flexible microfluidicdevices for colorimetric analysis of sweat on the skin (a) and undermechanical diction of bending (b). (c) Top view illustration ofmicrofluidic device with colorimetric assays and reference marker. d)Exploded view illustration of a device and its interface with skin.

FIG. 59. (a) Schematic illustration of device with color referencemarkers of pH, creatinine and urea, and number for indicating sweatcollection volume. (b)-(d) Color level of each concentration (top) andoptical images color development of assay chambers according to sampleconcentrations (top) of b) creatinine, c) urea, and d) pH.

FIG. 60. Sample analysis wells with overprinting colors (Yellow,Magenta, Cyan, Green) at different opacities (100, 90, 80, 75, 50, 40,30, 25, 20, 10) and two control points per pattern. Control points haveno printing, but contain printed overlay material (PET) to eliminatepath length variations. Duplication of each row eliminates channelheight variation. Colorimetric assay is silver chloranilate for a 75 mMconcentration test solution.

FIG. 61. Facet plot of the color Green of measured chroma values versusconcentration (known). The facets represent the different opacities. Theoverprints were made via laser printer.

FIG. 62. Microfluidic channels forming a “reservoir” that spatiallyholds 5 μL of fluid. When halfway full, the direction of fill changesthereby indicating both visually and via motion the current volume ofcollected fluid with respect to the total volume.

FIG. 63. A representative device showing a network of channel“reservoirs” that hold a larger volume of collected sweat with a“digital” indication of the total volume of collected sweat.

FIG. 64. (A) Sweat collection volume for one adult volunteer over 9 daysfor both the left and right arms. (B) Comparison of epifluidic deviceand MACRODUCT® sweat collection volumes for three adult volunteers. (C)Chloride concentration for collected sweat for two adult volunteers.

FIG. 65. (A) Optical image of an epidermal collection device withintegrated chloride colorimetric assay reservoirs. (B) The colorimetricassay increases in color intensity (violet) with increasing chlorideconcentration. When captured using a smartphone camera, the colorprovides a quantitative analysis of chloride levels.

FIG. 66. (A) Volume of collected sweat over a 5-day trial for a singleadult volunteer using a collection device with integrated analysis. (b)Comparison of colorimetric assay performance to CHLOROCHEK® measurementsover a 4-day trial for a single adult volunteer.

FIG. 67. a) Schematic illustration of mounting position of sweatcollection device on the body at forearm and type of exercise. b)Optical image of microfluidic device spotted with blue dye that mixeswith sweat. The extent of blue dye in the channel during sweat providesa measure of total sweat volume at any given instant in time. c)Correlation of sweat collection for a microfluidic device from theanterior forearm versus the normalized total body loss (based on initialweigh-in and final weigh-out with no fluid intake or restroom use duringexercise). d) Correlation of sweat collection for a microfluidic deviceversus an absorbent patch. e) Cumulative local sweat loss versus timemeasured from the forearm with a microfluidic device during exercise,while at rest, and during a subsequent exercise session.

FIG. 68. Human trials. (A) Photograph of a subject adorning the wirelessbattery-free hybrid sensor system. (B) Reading distance of device withlarge NFC antenna. (C) Image of complete system captured after a bout ofcycling by a subject. Real-time wirelessly acquired sweat concentrationlevels for (D) lactate and (E) glucose. (F) Image of complete systemcaptured after a bout of cycling by a subject. Real-time wirelesslyacquired sweat concentration levels for (G) lactate and (H) glucose. (I)Correlation of data acquired from biofuel cell-based glucose and lactatesweat sensors with that acquired from blood glucose and lactate meterover a period of two days for subject #1. (D, E, G and H) Blue regionrepresents no sweat while green indicates sweating of the humansubjects.

FIG. 69. Correlation of data acquired from biofuel cell-based glucoseand lactate sweat sensors with that acquired from blood glucose andlactate meter over a period of two days for (A) subject #2 and (B)subject #3.

FIG. 70. (A) Comparison of signal of fresh, unused glucose sensor(black) with that obtained from one after two-day human trial (red) whenexposed to 300 μM glucose solution. (B) Comparison of signal of fresh,unused lactate sensor (black) with that obtained from one after two-dayhuman trial (red) when exposed to 10 mM lactate solution.

FIG. 71. Human tests a: Various location on which the device is placedfor the human tests, b: The sauna environment for thermal sweat test, c:The gym environment for exercise sweat test, d and e: The comparison ofsweat excretion rate and sweat chloride concentration at running andsauna conditions with subject #1 and subject #2, and f-i: The comparisonof sweat excretion rate and sweat chloride concentration at the devicelocation, placed on forehead and forearm with subject #3, subject #4,subject #5, and subject #6.

FIG. 72. Correlation of data acquired from biofuel cell-based glucoseand lactate sweat sensors with those acquired from blood glucose andlactate meters over a period of one day for (A) subject #1 and (B)subject #2.

DETAILED DESCRIPTION OF THE INVENTION

In general, the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to standard texts, journalreferences and contexts known to those skilled in the art. The followingdefinitions are provided to clarify their specific use in the context ofthe invention.

“Microfluidic device” refers to a system, device or device componentcontaining liquid constrained in at least one physical dimensiongenerally of the order of nanometers to millimeters, optionallynanometers to microns. Microfluidic devices may include structures forcollecting, extracting, transporting, storing, analyzing and/oroutputting fluids, including biofluids. In some embodiments, the liquidis constrained to a lateral dimension selected over the range of 1 nmand 1 cm, such as a lateral dimension (e.g., depth) selected over therange of 1 nm to 5 mm, 100 nm to 1000 μm or 500 nm to 100 μm, and alateral dimension (e.g., width) selected over the range of 1 nm to 1 cm,10 μm to 2 mm or 1 μm to 10 mm. In embodiments, an axial (e.g., flow)direction in a microfluidic system, device or device component can belong, for example on the order of meters, but will more commonly be 0.1cm to 100 cm or 1 cm to 50 cm. Microfluidics are distinguished hereinfrom macrofluidics. In some embodiments, the invention provides tissuemounted, optionally skin mounted, microfluidic devices. Microfluidicdevices of some embodiments are capable of determining the compositionof a biofluid such as sweat, for example, the presence, absence, and/oramount of one or more biomarkers, optionally as a function of time.Microfluidic devices of some embodiments are capable of determining oneor more physical parameters characteristics of a biofluid, such asamount, volume, release rate and/or absorption rate, optionally as afunction of time.

“Tissue-mounted” refers to systems, devices or device components havingat least one surface capable of being supported, directly or indirectly,by a tissue surface, for example in a configuration providing fluidiccommunication and/or conformal contact. Epidermal systems and devicesare a subset of tissue-mounted systems wherein the system, device ordevice component has at least one surface capable of being supported,directly or indirectly, by a surface of the skin, for example in aconfiguration providing fluidic communication and/or conformal contact.The invention provides tissue-mounted devices, such as epidermalsystems, capable of collection, storage, treatment, processing, handlingand/or analysis of biofluids such as sweat.

The expression “at least partially embedded in” refers to aconfiguration wherein an element, such as a microfluidic network orcomponent thereof, is at least partially, and optionally wholly,integrated on or within a layer and/or device component, such as asubstrate. In an embodiment, for example, “at least partially embeddedin” refers to a configuration wherein an embedded element, such as amicrofluidic element such as an inlet, outlet, passage, channel, and/orreservoir, at least partially comprises one or more surfaces, recessedfeatures, relief features or any combination thereof, within or on alayer or device component it is at least partially embedded in. In anembodiment, for example, “at least partially embedded in” refers to aconfiguration wherein an embedded element, such as an inlet, outlet,passage, channel, and/or reservoir, at least partially comprisesfeatures molded or embossed on or into a layer or device component it isat least partially embedded in. In an embodiment, for example, “at leastpartially embedded in” refers to a configuration wherein an embeddedelement, such as an inlet, outlet, passage, channel, and/or reservoir,at least partially comprises features at least partially comprisingsurfaces (e.g., top, bottom, walls, etc.) of a layer or device componentit is at least partially embedded. In an embodiment, for example, “atleast partially embedded in” refers to a configuration wherein anembedded element, such as an inlet, outlet, passage, channel, and/orreservoir, is at least partially covered or encapsulated by anotherdevice component, such as an top layer or barrier layer.

“Substrate” refers to a device component, such as a layer, having asurface that is capable of supporting, accommodating, embedding orotherwise integrating a structure, including a microfluidic structure,optical structure, electronic structure, thermal structure or anycombination of these. Substrates in some embodiments are capable ofsupporting, accommodating, embedding or otherwise integrating a devicecomponent such as microfluidic device component, optical devicecomponent, electronic device component, structural device component orany combination of these. In some embodiments, a substrate is capable ofat least partially forming an interface with the tissue of a subject,such as with the epidermis or other organ of a subject. In anembodiment, a substrate of the present devices, systems and methods is abiocompatible and/or bioinert material. In an embodiment, a substrate ofthe present devices, systems and methods is a polymer or elastomermaterial. Substrates of the invention include “functional substrates”which refers to a substrate component for a device having at least onefunction or purpose in addition to providing mechanical support for acomponent(s) disposed on or within the substrate such as a microfluidicfunctionality, a mechanical functionality, optical functionality or athermal functionality. A functional substrate may facilitate mechanical,thermal, chemical and/or electrical matching of the functional substrateand the skin of a subject such that the mechanical, thermal, chemicaland/or electrical properties of the functional substrate and the skinare within 20%, or 15%, or 10%, or 5% of one another. Devices andsystems of the invention may have more than one substrate, for example,such as embodiments having a bottom substrate capable of establishing aninterface with skin and an upper substrate layer, such as a barrierlayer providing an interface with an ambient environment. For example,the invention includes devices and systems having a multilayer geometryincluding a substrate and barrier layer.

In some embodiments, a substrate is mechanically matched to a tissue,such as mechanically matched to skin. In an embodiment, a mechanicallymatched substrate is optionally capable of providing an interface forestablishing fluid communication and/or conformal contact with a surfaceof the tissue, such as skin. Devices and methods of certain embodimentsincorporate substrates comprising soft materials, for example exhibitingflexibility and/or stretchability, such as polymeric and/or elastomericmaterials. In an embodiment, a mechanically matched substrate has amodulus less than or equal to 100 MPa, and optionally for someembodiments less than or equal to 10 MPa, and optionally for someembodiments, less than or equal to 1 MPa. In an embodiment, amechanically matched substrate has a thickness less than or equal to 0.5mm, and optionally for some embodiments, less than or equal to 1 cm, andoptionally for some embodiments, less than or equal to 3 mm. In anembodiment, a mechanically matched substrate has a bending stiffnessless than or equal to 1 nN m, optionally less than or equal to 0.5 nN m.

“Polymer” refers to a macromolecule composed of repeating structuralunits connected by covalent chemical bonds or the polymerization productof one or more monomers, often characterized by a high molecular weight.The term polymer includes homopolymers, or polymers consistingessentially of a single repeating monomer subunit. The term polymer alsoincludes copolymers, or polymers consisting essentially of two or moremonomer subunits, such as random, block, alternating, segmented,grafted, tapered and other copolymers. Useful polymers include organicpolymers or inorganic polymers that may be in amorphous, semi-amorphous,crystalline or partially crystalline states. Crosslinked polymers havinglinked monomer chains are particularly useful for some applications.Polymers useable in the methods, devices and components disclosedinclude, but are not limited to, plastics, elastomers, thermoplasticelastomers, elastoplastics, thermoplastics and acrylates. Exemplarypolymers include, but are not limited to, acetal polymers, biodegradablepolymers, cellulosic polymers, fluoropolymers, nylons, polyacrylonitrilepolymers, polyamide-imide polymers, polyimides, polyarylates,polybenzimidazole, polybutylene, polycarbonate, polyesters,polyetherimide, polyethylene, polyethylene copolymers and modifiedpolyethylenes, polyketones, poly(methyl methacrylate),polymethylpentene, polyphenylene oxides and polyphenylene sulfides,polyphthalamide, polypropylene, polyurethanes, styrenic resins,sulfone-based resins, vinyl-based resins, rubber (including naturalrubber, styrene-butadiene, polybutadiene, neoprene, ethylene-propylene,butyl, nitrile, silicones), acrylic, nylon, polycarbonate, polyester,polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyolefinor any combinations of these.

“Elastomer” refers to a polymeric material which can be stretched ordeformed and returned to its original shape without substantialpermanent deformation. Elastomers commonly undergo substantially elasticdeformations. Useful elastomers include those comprising polymers,copolymers, composite materials or mixtures of polymers and copolymers.Elastomeric layer refers to a layer comprising at least one elastomer.Elastomeric layers may also include dopants and other non-elastomericmaterials. Useful elastomers include, but are not limited to,thermoplastic elastomers, styrenic materials, olefinic materials,polyolefin, polyurethane thermoplastic elastomers, polyamides, syntheticrubbers, PDMS, polybutadiene, polyisobutylene,poly(styrene-butadiene-styrene), polyurethanes, polychloroprene andsilicones. Exemplary elastomers include, but are not limited to siliconcontaining polymers such as polysiloxanes including poly(dimethylsiloxane) (i.e. PDMS and h-PDMS), poly(methyl siloxane), partiallyalkylated poly(methyl siloxane), poly(alkyl methyl siloxane) andpoly(phenyl methyl siloxane), silicon modified elastomers, thermoplasticelastomers, styrenic materials, olefinic materials, polyolefin,polyurethane thermoplastic elastomers, polyamides, synthetic rubbers,polyisobutylene, poly(styrene-butadiene-styrene), polyurethanes,polychloroprene and silicones. In an embodiment, a polymer is anelastomer.

“Conformable” refers to a device, material or substrate which has abending stiffness that is sufficiently low to allow the device, materialor substrate to adopt a desired contour profile, for example a contourprofile allowing for conformal contact with a surface characterized by asurface topography comprising recessed and/or relief features. Incertain embodiments, a desired contour profile is that of tissue, suchas skin.

“Conformal contact” refers to contact established between a device and areceiving surface. In one aspect, conformal contact involves amacroscopic adaptation of one or more surfaces (e.g., contact surfaces)of a device to the overall shape of a surface. In another aspect,conformal contact involves a microscopic adaptation of one or moresurfaces (e.g., contact surfaces) of a device to a surface resulting inan intimate contact substantially free of voids. In an embodiment,conformal contact involves adaptation of a contact surface(s) of thedevice to a receiving surface(s) such that intimate contact is achieved,for example, wherein less than 20% of the surface area of a contactsurface of the device does not physically contact the receiving surface,or optionally less than 10% of a contact surface of the device does notphysically contact the receiving surface, or optionally less than 5% ofa contact surface of the device does not physically contact thereceiving surface. In some embodiments, devices of the invention arecapable of establishing conformal contact with tissue of a subject, suchas a portion of the skin of a subject.

“Young's modulus” is a mechanical property of a material, device orlayer which refers to the ratio of stress to strain for a givensubstance. Young's modulus may be provided by the expression:

$\begin{matrix}{{E = {\frac{({stress})}{({strain})} = {( \frac{L_{0}}{\Delta\; L} )( \frac{F}{A} )}}},} & (I)\end{matrix}$where E is Young's modulus, L₀ is the equilibrium length, ΔL is thelength change under the applied stress, F is the force applied, and A isthe area over which the force is applied. Young's modulus may also beexpressed in terms of Lame constants via the equation:

$\begin{matrix}{{E = \frac{\mu( {{3\lambda} + {2\mu}} )}{\lambda + \mu}},} & ({II})\end{matrix}$where λ and μ are Lame constants. High Young's modulus (or “highmodulus”) and low Young's modulus (or “low modulus”) are relativedescriptors of the magnitude of Young's modulus in a given material,layer or device. In some embodiments, a high Young's modulus is largerthan a low Young's modulus, preferably about 10 times larger for someapplications, more preferably about 100 times larger for otherapplications, and even more preferably about 1000 times larger for yetother applications. In an embodiment, a low modulus layer has a Young'smodulus less than 100 MPa, optionally less than 10 MPa, and optionally aYoung's modulus selected from the range of 0.1 MPa to 50 MPa. In anembodiment, a high modulus layer has a Young's modulus greater than 100MPa, optionally greater than 10 GPa, and optionally a Young's modulusselected from the range of 1 GPa to 100 GPa. In an embodiment, a deviceof the invention has one or more components having a low Young'smodulus. In an embodiment, a device of the invention has an overall lowYoung's modulus.

“Sensing” refers to an action of detecting the presence, absence,amount, magnitude and/or intensity of one or more physical and/orchemical properties or characteristics. Sensor refers to a device orcomponent thereof that is capable of sensing. Useful device componentsfor sensing include, but are not limited to electrode elements, chemicalor biological sensor elements, pH sensors, colorimetric sensors,electrochemical sensors, temperature sensors, strain sensors, mechanicalsensors, position sensors, optical sensors and capacitive sensors.

“Actuating” refers to an action of acting on, stimulating, controlling,or otherwise affecting a structure, material or device component.Actuator refers to a device or component thereof that is capable ofactuating. Useful device components for actuating include, but are notlimited to, electrode elements, electromagnetic radiation emittingelements, light emitting diodes, lasers, magnetic elements, acousticelements, piezoelectric elements, chemical elements, biologicalelements, and heating elements.

“Bending stiffness” is a mechanical property of a material, device orlayer describing the resistance of the material, device or layer to anapplied bending moment. Generally, bending stiffness is defined as theproduct of the modulus and area moment of inertia of the material,device or layer. A material having an inhomogeneous bending stiffnessmay optionally be described in terms of a “bulk” or “average” bendingstiffness for the entire layer of material.

The terms “flexible” and “bendable” are used synonymously in the presentdescription and refer to the ability of a material, structure, device ordevice component to be deformed into a curved or bent shape withoutundergoing a transformation that introduces significant strain, such asstrain characterizing the failure point of a material, structure, deviceor device component. In an exemplary embodiment, a flexible material,structure, device or device component may be deformed into a curvedshape without introducing strain larger than or equal to 5%, for someapplications larger than or equal to 1%, and for yet other applicationslarger than or equal to 0.5% in strain-sensitive regions. A used herein,some, but not necessarily all, flexible structures are also stretchable.A variety of properties provide flexible structures (e.g., devicecomponents) of the invention, including materials properties such as alow modulus, bending stiffness and flexural rigidity; physicaldimensions such as small average thickness (e.g., less than 10000microns, optionally less than 1000 microns and optionally less than 100micron) and device geometries such as thin film and mesh geometries.

“Stretchable” refers to the ability of a material, structure, device ordevice component to be strained without undergoing fracture. In anexemplary embodiment, a stretchable material, structure, device ordevice component may undergo strain larger than 0.5% without fracturing,for some applications strain larger than 1% without fracturing and foryet other applications strain larger than 3% without fracturing. A usedherein, stretchable structures may also be flexible. Some stretchablestructures (e.g., device components) are engineered to be able toundergo compression, elongation and/or twisting so as to be able todeform (and optionally operate) without fracturing. Stretchablestructures include structures comprising stretchable materials, such aselastomers; and bent, coiled or serpentine structures capable ofelongation, compression and/or twisting motion.

Devices of the present invention may optionally include one or morebarrier layers. As used herein “barrier layer” refers to a devicecomponent spatially separating two or more other device components orspatially separating a device component from a structure, material,fluid or ambient environment external to the device. In one embodiment,a barrier layer encapsulates one or more device components. Inembodiments, a barrier layer separates one or more device componentsfrom an aqueous solution, a biological tissue and/or a biologicalenvironment. In some embodiments, a barrier layer is a passive devicecomponent. In some embodiments, a barrier layer is a functional, butnon-active, device component. In a specific embodiment, a barrier layeris a moisture barrier. As used herein, the term “moisture barrier”refers to a barrier layer which provides protection to other devicecomponents from bodily fluids, ionic solutions, water or other solvents.In one embodiment, a moisture barrier provides protection to an externalstructure, material or fluid, for example, by preventing leakage currentfrom escaping an encapsulated device component and reaching the externalstructure, material or fluid.

“Biofluid” refers to fluid generated by, extracted from or otherwisederived from the tissue of a subject, such as an organ of a subject.Biofluids include sweat, tears, saliva, gingival crevicular fluid,interstitial fluid, blood and combinations thereof.

As used herein, the term “fluidically connected” refers to theconfiguration of two or more components such that a fluid (e.g., a gasor a liquid) is capable of transport, flowing and/or diffusing from onecomponent to another component, without adversely impacting thefunctionality of each of the components. Components may be in fluidcommunication via one or more elements such as channels, valves, tubes,containment structures, reservoirs, pumps or any combinations of these.Components may be in fluid communication are in a direct fluidcommunication manner wherein fluid is capable of transport directly fromone component to another. Components may be in fluid communication in anindirect fluid communication manner wherein fluid is capable oftransport indirectly from one component to another via one or moreintermediate structures separating the components.

The term “operably connected” refers to a configuration of elements,wherein an action or reaction of one element affects another element,but in a manner that preserves each element's functionality. In anillustrative example, an electrochemical sensor operably connected to anelectronic device providing for wireless power harvesting refers to theability of the electrochemical sensor to be connected to the electronicdevice in such a way as to receive wireless power without adverselyimpacting the functionality of the electrochemical sensor and theelectronic device. In another illustrative example, a sensor (e.g.,capacitive sensor) operably connected to a microfluidic network refersto the sensor's ability to sense one or more parameters of a biofluid,or component thereof, which is being transported by the microfluidicnetwork, without adversely impacting the functionality of the sensor orof the microfluidic network. The connection may be by a direct physicalcontact between elements. The connection may be indirect, with anotherelement that indirectly connects the operably connected elements. Forexample, a capacitive sensor may be indirectly connected to themicrofluidic network, with a dieletric layer physically separating thesensor and the microfluidic network.

The terms “electrical contact” and “electronic contact” refers to theability of two or more materials and/or structures that are capable oftransferring charge between them, such as in the form of the transfer ofelectrons or ions. The terms “electrical contact” and “electroniccontact” may refer to a configuration of two or more components suchthat an electronic signal or charge carrier can be directly orindirectly transferred from one component to another. As used herein,the terms “electrical contact” and “electronic contact” include one wayand two way electrical communication. In some embodiments, components inelectrical contact or electronic contact are in indirect electricalcommunication wherein an electronic signal or charge carrier isindirectly transferred from one component to another via one or moreintermediate structures, such as circuit elements, separating thecomponents.

As used herein, the term “electrical load” may refer to voltage orcurrent applied to electrodes, sensors or other device components. Theterm “electrical response” or “electrical parameter” may refer to avoltage, current, or impedance response of the electrodes or sensors tothe electrical load. For example, applying a current between twoelectrodes (electrical load) may induce a voltage drop between the twoelectrodes (electrical response). The electrical load may be a DC or anAC load.

The term “BLE” refers to a Bluetooth low energy system.

The term “functionalized” may refer to modification of a material orlayer surface to add chemical, physical, electrical, optical orelectrochemical functionality. In an embodiment, biological molecules orreagents may be deposited onto an electrode in a process of forming anelectrochemical sensor.

The term “wet environment” may refer to the system being in ahigh-humidity environment or being at least partially surrounded by aliquid. The term “high-humidity” refers to the relative humidity of thesurroundings being >70%.

Provided herein are examples related to epidermal microfluidic systemsand methods, including device architectures, components specifications,for temporally resolved epidermal sampling, collection and sensing ofbiofluids (e.g., sweat) and complementary methods of making and usingthe devices. Relevant device parameters and ranges for enablingwell-defined temporal characterization of sweat including quantitativemeasurements of sweat rate, pressure and volume are described.

Other aspects include: Inlet, microfluidic network and CBV geometries,materials and dimensions for chrono-sampling; composite and multi-layerencapsulation and reinforcement strategies to mitigate fluid loss andaddress mechanical motion; microfluidic designs to address both high andlow sweat regimes; integration of active and passive components toadjust sweat flow (e.g., absorbents, heaters, etc.); re-usablemicrofluidic systems; underwater microfluidic systems; Fluid purge andreset functionality.

Also provided herein are Epidermal Sensing Systems for Optical Readout,Visualization and Analysis of Biofluids. Provided are sensing systemsand methods, including device architectures, components andspecifications for optical readout, visualization and analysis ofbiofluids and components thereof (e.g., biomarkers), as well ascomplementary methods of making and using the devices.

Referring to the figures presented herein, a microfluidic system 10 maycomprise a flexible substrate 20 and at least two microfluidic networks30 40, with each network comprising a microfluidic inlet conduit network40, a biofluid inlet 50 and a plurality of reservoir chambers 60. Aplurality of capillary burst valves 70 may be in fluidic contact themicrofluidic conduit network, with a valve positioned betweenfluidically adjacent reservoir chambers. To assist in fluid fillingobservation, colorimetric sensors 80 may be positioned in reservoirchambers. Microfluidic outlet conduit 90 may connect to the chamber 60,including to relieve gas back pressure to the chamber, thereby improvingcontrolled and accurate filling of chambers.

Referring particularly to FIG. 7, a plurality of reservoir networks 100may be embedded in flexible substrate 20. Each reservoir network 100 maycomprise reservoir chambers 110, biofluid inlet 120, capillary burstvalve 130, and outlet 140 fluidically connected to the reservoirchamber. A colorimetric sensor or fluid indicator 145 may assist withvisualization of fluid filling in the network. In a similar manner, acolor indicator strip 150 may be positioned between fluidically adjacentreservoir chambers 110 (see, e.g., FIG. 12).

Other examples of sensors beyond color-changing sensors, include sensorshaving electric or electronic components, including the sensors 160illustrated in FIG. 20. As desired, a capping layer 170 may be providedon the top and/or skin-facing surface of the system. As desired, anadhesive layer 200 may help facilitate intimate and reliable skinsurface contact. The capping layer may be patterned, such as with relief172 and/or recess 174 features, including a recess feature that may be apassage to facilitate biofluid entry.

A biofluid gelling additive 210 may be provided in the microfluidicnetwork, such as to increase viscosity (FIG. 17). An absorbent(s) 270may be used in the microfluidic network.

FIGS. 29-30 illustrate use of patterned gratings 300 and indicators 310to help facilitate observation of biofluid in the system.

FIG. 31 illustrates expunge port that may be utilized to remove biofluidfrom the reservoir chamber. The expunge port may comprise two outlets322 324.

A collection layer 400 may promote or help facilitate biofluid transportfrom the skin (FIG. 37). Any of the systems may have a heater 500 tohelp regulate biofluid availability (FIG. 19) and/or may be incorporatedinto a glove 540. High sensitivity electrodes 510 may measure change inan electrical parameter, thereby measuring a biofluid parameter ofinterest. A wireless communication device 520 may facilitatetransmission of information wirelessly, including to a receiver 525,such as a hand-held device (FIG. 21). A protective layer 530 may beembedded in or supported by the flexible substrate (FIG. 15). FIG. 21illustrates that any of the systems may have a disposable portion 560and a reusable portion 560, such as corresponding to the fluidics andelectronics components, for example (FIG. 21), by releasable couplingelement (580).

Example 1: Capillary Bursting Valves for Sequential Sampling(62/514,489)

Thin, Soft Microfluidic Devices for Chrono-Sampling of Sweat: The thingeometries and soft mechanics of these devices allows their intimate,comfortable bonding to the skin for the purpose of collecting,manipulating, analyzing, and storing sweat, captured in a sequentialmanner. An example device shown in FIG. 1 has a circular overallgeometry with a diameter of 3 cm. The radial construction facilitatesthe use of centrifugation techniques for collection of sweat afterremoving the device from the skin, as described subsequently. Theexample design involves two layers of poly(dimethylsiloxane) (PDMS)supported on a medical-grade acrylic adhesive film for bonding to theskin. The first layer defines a network of microfluidic channels (e.g.,400 μm thickness; channel widths and heights are 200 and 300 μm,respectively, for example) and the CBVs (designs described next). Thesecond serves as a capping layer (e.g., 200 μm thickness; inlet), andthe third (e.g., 50 μm thickness) establishes adhesion to the skin anddefines openings (e.g., 2 mm diameter) from which sweat can enter themicrofluidic system (e.g., 1 mm diameter, inlet; FIG. 1). The exemplarystructure in FIG. 1 comprises a network of microfluidic channels thatconnects to 12 separate chambers in parallel by bridging channels (FIG.1(c)). Each chamber connects to an outlet opening (e.g., 0.5 mmdiameter) designed to allow release of air that would otherwise betrapped in the chamber and serve as a source of backpressure tofrustrate the filling of sweat into the chamber. In vitro tests usingdyed water illustrate the clockwise flow through this network (FIG.1(d)). PDMS may be used due to its dimensional stability in water,materials biocompatibility, low modulus, elastic mechanical properties,and compatibility with simple molding and bonding processes forfabrication. Amino acids, glucose, and pyruvate may exhibit lowabsorption into PDMS. Certain chemistries, including certain vitaminsand hormones, have comparatively high absorption, but theirconcentrations are generally not crucial to analysis of sweat. Testingindicates an absence of chemical contamination from the PDMS and theadhesive layer in analysis of biomarkers of interest in sweat. Thesesame results suggest a minor (≈10%) decrease in glucose concentration,possibly due to slight absorption into the constituent materials of thedevice.

Principle and Design of the Capillary Bursting Valves for SequentialSampling: The CBVs block flows at pressures lower than theircharacteristic bursting pressures (BPs). When liquid in a singleconnected channel encounters two separate CBVs with different BPs, atsufficient pressures, the flow will proceed first through the valve withlower BP. In this way, locating two CBVs with different BPs near theintersection between two channels allows control of the direction offlow. The Young-Laplace equation gives the BP in a rectangular channelas equation (1)

$\begin{matrix}{{BP} = {{- 2}{\sigma( {\frac{\cos\;\theta_{I}^{*}}{w} + \frac{\cos\;\theta_{A}}{h}} )}}} & (1)\end{matrix}$where σ is the surface tension of liquid, θ_(A) is the contact angle ofthe channel, θ_(l)* is the min[δ_(A)+β; 180°], β is the diverging angleof the channel, w and h are the width and the height of the divergingsection, respectively.

For hydrophobic materials at high diverging angles, the BP increaseswith decreasing b and h. Each unit cell of the devices described hereincludes three CBVs, a collection chamber, an extraction chamber and asampling outlet (FIG. 2(a)). In one embodiment, the first two CBVs,denoted #1 and #2, have diverging angles of 13° and 90°, respectively,and widths of 200 μm. The third CBV, i.e., #3, has a diverging angle of120 Åã and a width of 50 μm (FIG. 2(b)). The heights of these valves are300 μm. According to Equation (1), the contact angle of the channelsurfaces affects the BP. PDMS, which is naturally hydrophobic, becomeshydrophilic after exposure to oxygen plasma for the purpose ofactivating the surfaces to enable bonding. The hydrophobicity recoversafter ≈24 h, to reach a constant, time-independent contact angle of107°. Based on this parameter, the computed BPs for CBVs #1, #2, and #3are 498.9 (BP #1), 881.7 (BP #2), and 3035.7 Pa (BP #3), respectively.Experimentally measured values are somewhat lower than these estimates,mainly due to imperfections in the fabrication and, in particular,diverging angles that are slightly smaller than the design values, asshown in the SEM images in FIG. 2(a). For example, in CBV #2 and #3, thesharp edges where the straight channel and the diverging sectionintersect are somewhat rounded, with radii of curvature of ≈35 and 27μm, respectively. Liquid that initially arrives at CBVs #1 and #2encounters them in their closed states (FIG. 2e (i)). Upon reaching orexceeding BP #1, CBV #1 opens to allow flow into the chamber (FIG. 2e(ii)). After filling this chamber, the liquid flow bursts CBV #2 atsufficient pressure (BP of CBV #2 is lower than that of CBV #3) (FIG. 2e(iii)). By this process, all 12 chambers fill in a sequential manner,for flows that involve pressures larger than BP #2. Due to the pressuredrop along the channel, the required pressure to fill the whole chambersis ≈1000 Pa and is higher than BP #2. For constant flow rate, thiseffect translates to time-sequenced sampling, or chrono-sampling. Afteruse, the device can be removed from the skin and then inserted into acentrifuge (5000 rpm) to open CBV #3, thereby moving liquid from each ofthe storage chambers into corresponding extraction chambers tofacilitate recovery for lab analysis (FIG. 2e (iv)). The designs of theCBVs ensure that pressures generated by the sweat glands exceed BP #1and BP #2, thereby allowing complete filling of the associated chambers,and that centrifugal pressures exceed BP #3. The pressure generated bythe sweat glands exceeds BP #3, but it does not burst this valve untilafter filling the chamber.

Sweat Sample Extraction Process: After collecting sweat, a centrifuge isused to recover the sweat (FIG. 3). During the centrifuge, the sweat inthe chamber is moved to extraction chamber and each sweat from differenttime is separated. We can extract the sweat by simple pipetting. Thisdevice contains about 3 pL from each chamber, for example.

Various Size and Chamber Number of chrono-sampling device: Microfluidicsystems may have various dimensions (e.g., 1 cm-5 cm in diameter) withdifferent numbers (e.g., 1-24) of chambers, sizes of chambers, andshapes of chambers for chrono-sampling devices (FIG. 4). Microfluidicsystem design may be tuned to the application. The small devices may beused for short-term sweating and the large device may be used forlong-term exercise. The volume of chamber may be expanded to 100 uL forcomplex in vitro analysis. Two microfluidic networks may be incorporatedin one microfluidic system.

Designs for Accurate Measurement of Sweat Loss: As a detail, a singlemicrofluidic system may be used for high biofluid loss regime and theother is used for low loss regime (FIG. 5). Each of two differentmicrofluidic networks is used for a different measuring regime. In theleft device, by changing the collecting area, the outer unit collectssweat rapidly. The area of opening in adhesive is 4 times bigger inoutside. Therefore, the sweat fills four times faster in outside device.Therefore, the outside device can be used as minute hand of a watch.Using both units we can calculate sweat rate more accurately. See alsoAddendum A.

Thin, Soft Microfluidic Devices for Measuring Pressure from Sweat Gland:Sweat is coming from sweat gland by the pressure generated from osmoticpressure. The pressure can be derived from following equation.P=σRTΔCwhere σ is the osmotic reflection coefficient, R is the gas constant, Tis the temperature of the body, and ΔC is the difference inconcentration between plasma and sweat often represented in terms ofosmolality. For example, the device is attached to the skin and sweatgland is connected to the microfluidic channel with capillary burstingvalve (FIG. 6). When sweat is coming from the sweat gland, if thepressure from sweat gland is higher than the bursting pressure of valve,it will burst in the chamber. The example device is composed of threelayers; capping layer, microfluidic channel layer and adhesive layerwith opening of sweat. For the fabrication of the device,photolithographic process is performed.

Serial CBVs with Different Bursting Pressure:

In FIG. 7, a microfluidic system has 12 values with different burstingpressure accomplished by changing the valve size from 120 μm to 10 μm(FIG. 7). The bursting pressure increases. If the sweat has pressurehigher than such valves it will burst and stop at certain valve. Tovisualize the bursting of valve, cobalt chloride is used in the chamber.When there is bursting the color is changed to red. The device maydetect from 1.2 kPa to 6.5 kPa, for example. In vitro tests measure thebursting pressure of the device and numerical analysis yielded wellmatched values.

Calculation of bursting pressure in vitro and in computing: For thecalculation of bursting pressure of CBVs (equation 1), we have to knowthe static and advancing contact angle of the surface of the device. Wemeasure the values by using contact angle goniometer (FIG. 8). Also, therounding edge of the CBVs affects to the bursting pressure. We measuredthe rounding edge using SEM image. Finally, we measure the burstingpressure using pressure generator and numerical calculation, asdescribed above for Eq. (1).

In situ Sweat Pressure Measurement: FIG. 9 shows the pressure measuredin exercising and thermal exposure conditions using the microfluidicsystem. The overall pressure value is measured as 1-2 kPa, in thisexample, which is smaller than the test using sweat inducer andmicropipette. This may be the first time sweat pressure at normalsweating condition has been measured. The pressure at exercising ishigher than thermal exposure. The pressure is proportional to skintemperature and concentration difference between sweat and plasma.Assuming that skin temperature is not different from each condition, theconcentration difference in exercising condition is larger than thermalexposure. The sodium concentration appears higher in the exercisecondition. From these results, it may be expected that sweat pressuremay allow determining body conditions like sweat concentration so on.

Colorimetric detection of chloride using silver chloranilate andmicrofluidic device: For the in situ analysis of sweat, colorimetricsensors may be used. Colorimetric may be read by naked eye or camera insmartphone. For the detection of chloride, silver chloranilate (SCL) maybe used (FIG. 10). SCL may be mixed with pHEMA(Polyhydroxyethylmethacrylate) as a carrier. When sweat comes into thechamber it reacts with SCL and generates a colored ion. By detecting thecolor density, the chloride concentration may be estimated.

Equation two represents the chemistry of SCL. The SCL is slightlysoluble in water and reacts with chloride ions and hydrogen from water.The reaction generates silver chloride and acid chloranilate ion withpurple color. With sufficient amount of SCL in the chamber, therefore,the color density is proportional to the chloride concentration.C₆Ag₂Cl₂O₄+4Cl⁻+H⁺→2AgCl₂(s)+HC₆Cl₂O₄ ⁻(purple ion)  (2)

According to the CI concentration, the amount of colored ion is setwhich defines the color of the chamber. A pre-defined volume forconsistent color development is advantageous.

Color Changes and Calibration Markers: The concentration range of CI insweat is from 10 to 100 mM. The microfluidic system is tested withdifferent CI concentration from 10 to over 100 mM (e.g., 150 mM) todetermine limits. The color changed with CI concentration from 10 to 125mM continuously, which covers the chloride concentration range in sweat.A simplified concentration to color level is derived (e.g., FIG. 12).Using the color value, we made a color calibration marker and located itat each sides of the chamber.

Detection using Color Marker: This microfluidic system may be used inreal world applications like gym and jogging conditions. By naked eye,the color level is compared to the calibration marker to see the CIconcentration roughly. By a smartphone app, the CI concentration isdetermined more accurately. For example, after taking picture, the appanalyzes color level using color meter in the app (FIG. 12). Afterextracting the color level in the calibration marker and chamber, theyare compared the CI concertation is determined. In FIG. 12, theconcentration value is 55.5 mM.

Accuracy of CI Detection: To test accuracy of chloride concentrationdetection in vitro the chamber of the microfluidic system is filled withknown a concentration and the color level in the chamber is analyzed andcompared to calibration indicator strip (FIG. 13). In all concentrationlevels, the concentration values are well matched to the calibrationmarker. In the in vivo test, the concentration according to color in thechamber with collected sweat is compared to results using lab sweatanalysis. In the first trial, the device measures a 65 mM chlorideconcentration, which is 8.1 mM higher than laboratory results. In thesecond test, the measured concentration is 6.8 mM lower than laboratoryresults.

Applications: The ability to collect sweat non-invasively, andlongitudinally has significant implications in human health tracking inboth normal and diseased states. Conventional technologies do notperform this task. For health tracking during physical activity,embodiments of the presently disclosed microfluidic systems and methodsprovide quantified feedback on sweat loss. This may serve as an earlywarning system of dehydration. Conventionally, the ability to assessvolume loss depends on clinical signs (e.g. dry mucous membranes,delayed capillary refill) that are often lagging. Also, embodiments ofthe presently disclosed microfluidic systems and methods may be used toassess the sweat response of an individual in the setting of physicalactivity. Individuals with greater athletic ability are capable ofdissipating sweat to maintain core body temperature with more efficiencythan non-athletes. embodiments of the presently disclosed microfluidicsystems and methods create novel metrics of athletic performance byquantifying sweat response to physical activity and local skintemperature.

Beyond athletics, sweat loss has important clinical implications aswell. The assessment of sweat content is used for the diagnosis ofcystic fibrosis (CF). CF is the most common fatal genetic disorder inCaucasians. embodiments of the presently disclosed microfluidic systemsand methods provide an alternative advantageous diagnostic platform forthis disease. Conventionally, the sweat analysis for CF requiresexpensive equipment, specialized technician expertise, and lacksrepeatability (variations greater than 30% between hospitals). There arealso rare genetic diseases (e.g. hypohidrotic ectodermal dysplasia,ichthyotic diseases) where the sweat glands are dysfunctional orimpaired. These individuals are at great risk of fatal heat stroke. Theability to assess sweat rate and skin temperature may provide an earlywarning system for these vulnerable patients. The detection ofbiomarkers and electrolytes in sweat can be correlated to serumbiomarkers and electrolytes. Embodiments of the presently disclosedmicrofluidic systems and methods enable a new platform that allows forcontinuous, non-invasive assessment of body homeostasis, includingtracking sweat glucose to assess serum glucose for diabetics. Forpatients with significant needle aversion, venipuncture for basic labscan be very challenging. In pediatric patients, venipuncture can also bevery traumatic. Embodiments of the presently disclosed microfluidicsystems and methods sweat device are useful in collecting importantclinical data without the need for venipuncture.

Series of separated chambers chemically decoupled from each other hascapabilities to provide a reaction chambers for different biomarkerdetection and to analyze the biomarker in time-sequential method. Seriesof capillary bursting valves (CBVs) enables the device to collect thesweat in time sequence and to provide individual reaction chamberswithout cross contamination to other chambers (ref). Liquid flows to theroute with less fluidic resistance, low bursting pressure of CBV.Young-Laplace equation provides the bursting pressure (BP) of CBV in arectangular channel as described in Eq. (1) above.

$\begin{matrix}{{BP} = {{- 2}{\sigma( {\frac{\cos\;\theta_{I}^{*}}{w} + \frac{\cos\;\theta_{A}}{h}} )}}} & (1)\end{matrix}$

By locating CBVs with different bursting pressure, the microfluidicchannel controls the direction of the liquid in the channel. FIG. 14ashows a set of CBVs. From #1 to #4 and the bursting pressure increaseswith its number; CBV #1 and #2 has 300 μm wide channel with 13° and 90°of diverging outlet, respectively. CBV #3 and #4 has 200 μm and 50 μmwide channel with 90° and 120° of diverging outlet, respectively.Experimental results show the difference of bursting pressure from eachCBVs. The values were smaller than theoretical value and it is mainlydue to the round edge generation during fabrication process that leadsdiverging angles to be decreased than the design values. When sweatflows from inlet, it first reach to intersection composed of CBV #1, #2and #3. Sweat first burst CBV #1 and fills the chamber #1. Then, CBV #4with highest BP blocks the sweat flow and sweat burst chamber #2. Afterfilling chamber #1 and #2, sweat burst CBV #3 and flows to the nextchambers. FIG. 14d shows that the microfluidic device can sequentiallycollect liquid without undesired bursting of CBVs. The flow propertiesof the microchannels and geometry of the microfluidic channel preventunwanted mixing. Specifically, for channel dimensions of hundreds ofmicrometers and flow rates from sweat up to 1.0 μl min⁻¹ in the device,the laminar flow with low Reynolds numbers (<1) generated and mixingoccurs only by molecular diffusion. The small intersection area betweenthe chambers defined by width and height of inlet of the chamber is 0.18mm² also minimize the diffusion effect. The example illustratesoperation with water dyed using different colors and introduced in atime sequenced manner. Over relevant time scales (≈1 h) and temperatures(≈37° C.), diffusion occurs only within the connecting channel betweenwater with different colors that does not affect to the reaction in thechamber (FIG. 14e (iv)). The fourth chamber in this example isrelatively dark due to mixing of the red dye in the bridge channel withblue dye.

Example 2: Mitigating Inadvertent Water Transport in EpidermalMicrofluidic Sensors (U.S. Pat. App. 62/514,374)

The accuracy of sweat capture and analysis from athletes in hot climatesand/or for long durations may be challenged by the evaporation of sweatfrom the device during and after capturing the sweat. This may be causedby the use of polymer materials which are poor barriers to water vapor,and/or through vapor loss through the outlet(s), for example. Describedherein are systems and methods for mitigating water vapor loss fromepidermal microfluidic systems via polymers with high barrier propertiesand/or by augmenting the vapor pressure of the captured sweat viagelling agents.

Applications of the present systems and methods include: preventingwater loss via evaporation from epidermal microfluidic sensors; andpreventing water uptake into epidermal microfluidic sensors from theenvironment during aquatic exercise.

Advantages of the present systems and methods include: improvingaccuracy of epidermal microfluid sensors by reducing rate of evaporativewater loss; and Reducing water uptake from the environment duringaquatic exercise

In certain embodiments, biofluid and/or vapor loss or uptake fromepidermal microfluidic systems may be mitigated by having layers (e.g.,the substrate and/or capping layer) at least partially formed of one ormore thermoplastic elastomers (TPEs) with good water barrier propertiesand high-strain-to-failure. In certain embodiments, biofluid and/orvapor loss or uptake from epidermal microfluidic systems may bemitigated by having a thin, patterned, high-barrier capping lay on topof a PDMS-based substrate layer, for example, which includes amicrofluidic network and sensor(s). In certain embodiments, biofluidand/or vapor loss or uptake from epidermal microfluidic systems may bemitigated by including one or more gelling agents in the microfluidicsystem to augment the vapor pressure of the collected sweat. Biofluidand/or vapor loss or uptake from epidermal microfluidic systems may alsobe mitigated by any combination of the above described features andmethods.

FIG. 15 illustrates cross sections of certain embodiments of themicrofluidic systems disclosed herein. The data plot shows fluid lossdue to evaporation from the microfluidic systems as a function of time.The data shows that water may evaporate through PDMS and that a cappinglayer, including a PET capping layer for example, may reduce fluid loss.

Thin, Soft Microfluidic Devices for Sweat Capture and Analysis ofAquatic Athletes: The thin geometries and soft mechanics of thesemicrofluidic systems allows for intimate and comfortable adhering to theskin for the purpose of collecting, manipulating, analyzing, and/orstoring biofluid (e.g., sweat), captured from aquatic athletes. Themicrofluidic system may include two layers (e.g., substrate with themicrofluidic features and a protective or capping layer) of a styrenicblock copolymer (SBC) supported on an adhesive film (e.g., medical-gradeacrylic) for bonding to the skin. The SBC may bestyrene-ethylene-butadiene-styrene (SEBS), styrene-isoprene-styrene(SIS), or styrene-butadiene-styrene (SBS), for example. The styrenicblock copolymer may be an oleogel styrenic block copolymer. The styrenecomposition may be between 10-50% of the polymer. The mechanicalproperties (e.g., modulus and elongation to failure) of the SBC may beaugmented by the addition of an additive such as low molecular weighthydrocarbon (e.g., paraffin oil). For example, weight ratios of additive(e.g., paraffin oil) to the SBC may be from 1:1 to 3:1, for example.Furthermore, any of the layers may further include a tackifier, such asrosin gum, to improve the adhesion of the device layers to each other orto the adhesive. Example weight ratios of tackifier additive to SBCpolymer with an additive may be 0.5:1 to 4:1, for example. The firstlayer (e.g., substrate) may define a network of microfluidic channels,reservoir chambers, an inlet, an outlet, and capillary burst valves,such as illustrated in FIG. 15. For example, the channels may be 400 μmin thickness, and channel widths and heights may be 200 and 300 μm,respectively). The second layer may be a capping layer. For example, thecapping layer may be in 200 μm thickness and may have a biofluid inletaligned to the biofluid inlet of the first layer. The third layer may bean adhesive layer to establish adhesion to the skin and further defineopenings from which sweat can enter the microfluidic system. Forexample, the adhesive layer may be 50 μm in thickness and the openingsin the adhesive layer may be 2 mm in diameter. The microfluidic systemincludes a biofluid inlet, which may be 1 mm in diameter, for example.The microfluidic system includes reservoir chambers connected amicrofluidic outlet conduit network. The microfluidic outlet conduitnetwork includes an outlet, which may be 0.5 mm in diameter, forexample. The outlet is designed to allow release of air that wouldotherwise be trapped in the chamber and serve as a source ofbackpressure to frustrate the filling of sweat into the chamber. FIG. 15illustrates cross sections of certain embodiments of the microfluidicsystems disclosed herein. FIG. 15 further shows fluid loss from thedepicted microfluidic systems as a function of time. The data shows thatwater may evaporate through PDMS and that a PET capping layer, forexample, may reduce fluid loss.

Reliable sweat collection in aquatic settings or in arid climatesrequires constituent materials with excellent barrier properties toprevent contamination or to eliminate evaporative loss, respectively.Results of measurements of water transport through SIS membranes andwater absorption into bulk SIS appear in FIG. 16D. Less than 80 mg ofwater vapor passes through a 125 μm thick, 1.8 cm2 SIS membrane over 12days in a humid (>90%) environment (a permeability of 4.6×10-8g-m/mm2/hr/Pa). SIS at 37° C. water absorbs less than 1.5% of its weightover the same period. Comparisons of evaporative loss of water fromdevices constructed in SIS and PDMS highlight the importance of barrierproperties for collecting and storing sweat (FIG. 16). SIS devices withopen outlets can store sweat at 37° C. for 4 hours with less than 20%loss while PDMS devices of comparable geometry lose ˜100% within 3hours.

Example 3: Mitigating Loss of Collected Sweat Due to Mechanical Motionin Epidermal Microfluidic Sensors (U.S. App. No. 62/514,455)

The capture and analysis of sweat from athletes requires the storage ofsweat to be resistant to a variety of motions including bending,stretching, twisting, and compression. Sweat that is stored andcontained by capillary burst valves may be susceptible to prematurebursting if environmental forces cause the pressure in the chamber toincrease above the capillary burst pressure. Disclosed herein aresystems and methods to mitigate the bursting of outlet valves bymechanical reinforcement of the chamber ceilings and/or gelling agents,according to certain embodiments.

Applications of the present systems and methods include preventing lossof sweat in epidermal microfluidic sensors due to motion, twisting,bending, or compression

Advantages of the present systems and methods include improvingrobustness of epidermal microfluidic sensors against motion-inducedsweat loss

In certain embodiments, sweat loss from the microfluidic system due tomechanical forces may be mitigated by having a thin, patterned, highmodulus capping layer on top of a PDMS or styrenic block copolymer (SBC)substrate layer having a microfluidic network. In certain embodiments,sweat loss from the microfluidic system due to mechanical forces may bemitigated by including one or more gelling agents in the microfluidicsystem to augment the viscosity of collected sweat. Sweat loss from themicrofluidic system may also be mitigated with the combination of athin, patterned, high modulus capping layer on top of a PDMS or styrenicblock copolymer (SBC) substrate layer and gelling agents supported bythe microfluidic network.

FIG. 17 illustrates cross sections of certain embodiments of themicrofluidic systems disclosed herein. In particular, a viscositymodifier may be utilized to provide additional stability to the chamberand capillary burst valve (CBV). Examples of viscosity modifiers areprovided and the photograph shows without viscosity modifier andattendant unwanted ejection (left photo) and with a viscosity modifierwhere unwanted ejection is avoided (right photo), each under amechanical deformation.

Patterned rigid capping layer for strain localization: A thin, flexiblepolymer with high barrier properties may be laminated on top of sweatcollection chambers. The capping layer may be selectively removed (e.g.,etched) above non-essential areas (e.g., regions not corresponding tomicrochannels, reservoirs, capillary burst valves, inlets, outlets,and/or sensors) to localize strains to these non-essential regions andallow stretching and flexing of the microfluidic system. The cappinglayer may mechanically reinforce the sweat chamber ceiling, therebyreducing the effect of mechanical strains on the volume and pressureinside the chamber. Capping layer materials may include commerciallyavailable polyolefins (polyethylene, polypropylene and polyisobutylene),polyesters (polyethylene terephthalate and polyethylene naphthalate),fluorocarbons (polyvinylidene chloride and polytetrafluoroethylene),polyamides (nylon), and polyimides(poly-oxydiphenylene-pyromellitimide).

Gelling agents for increasing the viscosity of collected sweat: Gellingagents (also referred herein as a viscosity modifier) may includewater-soluble cellulose derivatives (e.g., methyl cellulose orhydroxypropyl methylcellulose). Gelling agents may be added to any offeatures of the microfluidic system, such as the biofluid collectingreservoir chambers. The gelling agents may be selected according totheir ability to absorb a large amount of water relative to their mass,while undergoing relatively small changes in their volume. During use,for example, sweat may enter reservoir chambers and mix with the gellingagent(s), as a result of which the viscosity of the sweat may increasewithout a large volumetric expansion. For example, at concentrationshigher than 1:5 by weight (cellulose to biofluid) the captured biofluidmay become a semi-solid gel. For example, the gelling agents may bedepositing using air brushing. Exemplary gelling agents may also furtherinclude one or more of agar, sodium alginate or any of a number of watersoluble polymers.

Viscosity modifier for stable CBVs: Increasing the viscosity of biofluidat collection chambers can selectively contain it and preventsinadvertent ejection from the chamber when pressed, twisted, orotherwise subjected to mechanical deformation. Various viscositymodifiers can be used, and by adding it to individual chambers andenables unimpeded operation of the surrounding network of channels andchambers.

Viscosity modifiers include Methyl cellulose, hydroxypropylmethylcellulose (HPMC), Agar, sodium alginate, or any number of watersoluble polymers.

Example 4: “Skin-Like” Wearable Sensor for Capturing and MeasuringInsensible Sweat Loss for Analysis (U.S. Pat. App. 62/514,546)

Systems for capturing and measuring insensible sweat loss (e.g. sweatloss not measurable by conventional methods such as absorbent pads)during resting offer the potential to enable analysis of temporalvariations in water loss and biomarkers. Current sensors that rely onabsorbent pads and microfluidics need a certain amount of sweat in asequential manner for analysis, and are not easy to use for subjects whoare in the situation where it is difficult to do exercise for sweating,e.g., infants, patients, etc. A thin and “skin-like” wearable wirelessdevise is described that bonds to the skin to allow for capturing anddetecting insensible sweat in an interconnected set of micropumps andelectronic systems. Embodiments of the device have two characteristics:one is that the device is able to measure insensible sweat loss withtime as signals of capacitance changes by high sensitive electrodes andsend the data via a wireless system with NFC chips, the other that thedevice is able to capture and collect the sweat pumped up from skin bytwo kinds of capillary forces for biomarker analysis. The systems alsocan be combined with a RF heater for the purpose of inducing sweat andcapturing the sweat more efficiency.

Described herein are methods and system for measuring temporal changesin insensible sweat loss during resting by a high sensitivity electrodewith water capturable absorbents. The described system can capturing andstore a small amount of biofluid such as sweat, tear, blood, etc., foranalysis. Further, the systems may induce sweat by a local heatingsystem without specific drugs such as pilocarpine or acetylcholine.

Described is a method for measuring insensible sweat rates by using ahigh sensitivity electrode with water absorbents and systems therefor.Also described is a method for capturing a small amount of biofluid byusing a microporous absorbent as a micropump and systems therefor. Insome embodiments, the described systems and methods provide a heater toinduce sweat without drugs and collecting the sweat for use indiagnosis.

The described systems and methods can capture and detect a small amountof insensible sweat using a water capturable absorbent and an electronicsystem. The technology consists of two specific characteristics: one isa high sensitivity electrode combined with water capturable absorbentwhich enable to detect a small amount of sweat loss and measure thesweat rate, the other is a micropump system formed from two kinds ofcapitally force which enable to capture insensible sweat from skin andstore it. The device provides the information about water loss andbiomarkers in insensible sweat inducing physiological or psychologicalstimulations, which is useful both for diagnosis and for basic sciencestudy in neuron science, sleeping study, etc.

A “Skin-like” Wearable Wireless Sensors for Capturing and MeasuringInsensible Sweat Loss for Analysis is described herein. The softmechanics, electronics, and hydrophilic microporous geometries of thedevices enable them to reliably fit to the skin and to capture anddetect the nanoliter volume of insensible sweat released from skinefficiency (FIG. 18a ) during resting. A representative device shown inFIG. 18b has a rectangular geometry (the height, width and length are930 μm, 2 cm and 4 cm) and two detection areas: one consists of aelectronic system for detecting and measuring the amount of insensiblesweat loss with time, and the other consists of microporous polymers forcapturing and collecting insensible sweat from skin to analyzebiomarkers. The device can be attached to any part of body. The wholedevice involves four layers, i.e., bottom layer that is a polyester meshhaving pores with diameter of 74 μm (the thickness of 90 μm; the widthand length are 1 cm and 1.5 cm) to absorb sweat from skin, the secondlayer that is a medical-grade silicon adhesive film with thickness of140 μm for supporting the bottom and next layers and bonding to theskin, the third layer that is a poly(dimethylsiloxane) (PDMS) withthickness of 400 μm to support electrodes and porous polymer in the twoareas, respectively, and the top layer that is a polyethylene film withthickness of 300 μm to prevent from gas evaporations.

The structure of the area for measuring sweat rate is shown in thebottom right of FIG. 18b . The amount of sweat loss can be estimatedfrom the amount of evaporated sweat captured on the electrodes (theheight, width and length are 80 μm, 70 μm and 1 cm). Insensible sweatevaporates immediately in a normal condition because the amount is verysmall. (FIG. 18a ). The first layer of a polyester mesh capture theinsensible sweat came from skin by capillary force. The mild hydrophobicpolyester can release the captured liquid easily. The vaporized sweatfrom the polyester is captured by the dried polyvinyl alcohol (PVA) gelinstalled between the electrodes, and induces capacitance changes in theelectrode. By measuring the variation of the capacitance with time, therate of the sweat evaporated from skin can be estimated. The dried PVAgel that has a hydrophilic microporous structure enables the electrodesto detect a small amount of vaporized insensible sweat and to preventfrom the pressure in the area from increasing too much. The temporalvariations in the signal of capacitance can be recorded on anapplication installed in a cell phone via wireless system with a NFCchip. From the analysis of the data, the amount of sweat loss with timefor an appropriate time period such as sleeping time can be estimated.

The structure of the area for capturing and collecting insensible sweatto analyze chloride concentration in the sweat is shown in the bottomleft of FIG. 18b . The first layer of the polymer mesh fixed on one sideby medical-grade silicon/acrylic adhesive film is attached to skin andabsorbs the small amount of insensible sweat directly by their capillaryforce in the same way to the area for measuring sweat rate. The otherside of the polyester mesh is attached to a dried PVA hydrogel withdiameter of 1 cm and thickness of 400 μm including a chloride assayreagent that is installed on the top layer of PDMS. The dried hydrogelis same material to that on the electrode in the other area and has aporous structure of nanometer to several micro miters. The narrower poresize and the hydrophilicity of the PVA enable it to get strongercapillary force compared with the polyester mesh. Because of thedifference of the capillary force, the liquid moves from the polymermesh to the dried PVA hydrogel like a tree trunk. The reagent of thechloride assay kit introduced in the PVA hydrogel react with thechloride in the sweat and its color changes to blue in dependence of thechloride concentration. From the amount of sweat loss measured from theother area and the intensity of the color, the concentration can becalculated.

Sweat inducing system with RF heaters for collecting sweat efficiency.Local heating under high humidity can induce sweat efficiency withoutdrugs. We propose a system combined a radio frequency (RF) heatingsystem and a sweat sensor for collecting sweat in a short time foranalysis of chloride, glucose, lactate acid, etc. A representativesystem is shown in FIG. 19. The RF heaters with magnet can be introducedinside a glove and can be moved to any parts using magnetic force fromoutside of the glove. When the skin is heated above 40° C., aheat-stimulated sweat begins to be induced. A RF heater is installed onone side of a PDMS film with thickness of 400 μm that has a magnet onthe other side. By placing two or three heaters inside of a disposableplastic glove, the hand can be heated under a high humidity condition.The heat-stimulated sweat induced by the heater can be captured by thedevice of insensible sweat for an analysis of biomarkers in the sweat.

Example 5: Device Fabrication

Fabrication of the microfluidic module: The fabrication process beginswith sequential cleaning of a 4″ silicon wafer with isopropyl alcohol,acetone, deionized water, and a final rinse with isopropyl alcohol.Next, spin coating a 15 μm thick film of photoresist (KMPR 1010;Microchem, MA, USA) following by baking at 110° C. for 5 min on a hotplate prepares the system for photolithographic patterning to define thegeometry of the microfluidics. Exposing the wafer to UV light through aphotomask mounted on the wafer, followed by baking at 110° C. for 3 minin a closed chamber, and then for 2 min in an open setup patterned thephotoresist. Immersing the substrate in developer solution (AZ 917 MIF;Integrated Micro Materials, TX, USA) completed the process.Subsequently, deep reactive ion etching (STS Pegasus ICP-DRIE, SPTSTechnologies Ltd.) created 600 μm deep micro patterned trenches in thesilicon wafer. Finally, spin coating poly(methylmethacrylate) (PMMA;Microchem, MA, USA) on the pattered silicon mold and baking at 180° C.for 3 min primed the mold to facilitate release of polydimethylsiloxane(PDMS; Sylgard 184, Dow corning, MI, USA) cast and cured on top, asdescribed next.

Dispersing 5 wt % white silicone (Reynolds Advanced Materials, IL, USA)into a transparent PDMS precursor (10:1, Sylgard 184) yielded a thickliquid cast onto the mold by spin coating at 200 rpm. Curing at 70° C.for 1 h yielded a 700 μm thick, soft, white microfluidic structure. Amechanical punch tool defined 1-mm diameter inlet holes for thecolorimetric channels and 3-mm diameter inlet holes for theelectrochemical chambers. Pouring PDMS (10:1) on a PMMA coated siliconwafer then spin casting at 400 rpm and curing at 70° C. for 1 h produceda uniform, 200 μm thick slab as a cap for the microfluidic platform. Anadditional layer of PDMS (60:1) spin cast at 1000 rpm and cured foranother 1 h at 70° C. formed a thin, tacky coating. Separately, acommercial laser printer (Konica Minolta C454 PS color, Tokyo, Japan)printed color reference markers on a 25 μm thick polyester (PET) film(FLX000464; FLEXcon, MA, USA), and a CO2 laser (Universal Laser Systems,AZ, USA) defined sweat inlet holes in a skin adhesive membrane (PC2723U,ScapaHealthcare). Assembly of the microfluidic patch involved placingthe colorimetric assays, electrochemical sensors, and neodymium magnets(D0105 Nickel; SuperMagnetMan, AL, USA) into their respective chambersand then laminating the sticky side of the capping layer onto the top ofthe microfluidic patch. Plasma treating a skin adhesive membrane, thecolor reference marker film and the microfluidic platform with ahandheld corona generator yielded hydrophilic surfaces that allowedefficient bonding of the stack to complete the fabrication.

Development of colorimetric assays for chloride and pH: The colorimetricchloride assay solution consisted of 50 mg of silver chloranilate (MPBioscience, CA, USA) dispersed in 200 μl of 2 wt %polyhydroxyethylmethacrylate (Sigma-Aldrich, MO, USA) methanolicsuspension. Drop-casting 0.5 μl delivered this chloride assay cocktailin the chambers designated for chloride sensing. Suspending 4 mL ofuniversal pH dye (Fisher Scientific, NH, USA), 274 mg of polyvinylchloride (M.W. ˜233,000; Sigma Aldrich, MO, USA), 635 μl ofo-nitrophenyloctylether (Sigma Aldrich, MO, USA) and 508 μl of Aliquat(Sigma Aldrich, MO, USA) in 10 ml of tetrahydrofuran (Sigma Aldrich, MO,USA) yielded the pH assay solution. Dip-coating filter papers (SigmaAldrich, MO, USA) in the pH cocktail for 10 s, and allowing them to dryat ambient conditions for 15 min formed the solid-state pH assay.Cutting the pH assay paper into circular pads using a metal punch(diameter, 2 mm) and placing them in each of the chambers designated forpH sensing completed the process.

Fabrication of biofuel cell-based electrochemical sensors for lactateand glucose: Electron beam evaporation (AJA International Inc., MA, USA)formed a thin film of chromium (thickness, 10 nm) as an adhesion layer,followed by a layer of gold (thickness, 100 nm) as a conductor on a75-μm thick sheet of polyimide (Argon Inc., CA, USA). A UV laser (LPKF,Germany) patterned the gold coated polyimide sheet to define thecircular current collector, serpentine interconnects, and contact pads.The first step in realizing a biofuel cell-based lactate sensor involvedpunching out circular pads (diameter, 2 mm) of CNT paper (Thin FilmBA-01-145; NanoTechLabs, NC, USA). Coating with 2 μl of 0.1 Mtetrathiafulvalene (Sigma Aldrich, MO, USA) solution prepared inacetone/ethanol (1:9 v/v) and 4 μl of lactate oxidase (Toyobo Chemicals,Japan), and allowing them to dry, yielded enzyme functionalized CNTpads. The enzyme solution resulted from dispersing the enzyme (60 mg/ml)in 0.1 M phosphate buffer containing 0.25 wt % glutaraldehyde (SigmaAldrich, MO, USA). Subsequently, drop-casting and drying 2 μl ofchitosan (CAS Number 9012-76-4; Sigma Aldrich, MO, USA) suspensionprepared in 0.1 M acetic acid onto each pad formed a chitosan-basedmembrane. Dipping the dried pads into the chitosan solution for 5 s andthen allowing to dry resulted in an additional chitosan membrane.Finally, dipping the pads for 5 s in 3 wt % polyvinyl chloride (PVC)(CAS Number 9002-86-2; Sigma Aldrich, MO, USA) suspension intetrahydrofuran, and thoroughly air drying them formed the outer layerof PVC membrane. Conductive silver glue then bonded the pads to the goldcurrent collectors to complete the anode functionalization process. Thecathode for the lactate sensor resulted from drop casting 15 μl of 10mg/ml platinum black (Sigma Aldrich, MO, USA) suspension prepared indeionized water, followed by applying 1 μl of Nafion® 117 solution(Sigma Aldrich, MO, USA), onto the cathode designated gold currentcollector. Storing the sensors at 40 C for at least 1 week before useallowed the chitosan and PVC membranes to stabilize. Fabrication ofbiofuel-cell based glucose sensors involved steps similar to thosediscussed for the lactate sensor with some modifications. The processbegan with drop-casting 1 μl of 0.1 M tetrathiafulvalene solution ontoCNT pads. Separately, preparing a 40 mg/ml solution of glucose oxidasein 0.1 M phosphate buffer containing 10 mg/ml bovine serum albumin(Sigma Aldrich, MO, USA) and a 1 wt % suspension of Nafion® in 0.1 Mphosphate buffer and then mixing of the two suspensions in equal volumesyielded the enzyme coating suspension. Application of 2 μl of the enzymecoating suspension functionalized the tetrathiafulvalene coated CNTpads. Conductive silver glue bonded the pads to the gold currentcollectors to complete the anode. The glucose sensor cathode resultedfrom preparing a 10 mg/ml suspension of 10% platinum on carbon (SigmaAldrich, MO, USA) in a 2 wt % ethanolic suspension of Nafion® followedby casting 5 μl of the suspension on each current collector. Storing thesensors at 40° C. for at least 1 week before use allowed the Nafion®membrane to equilibrate. Both the lactate and glucose sensors werestable for at least 6 months when store at 40 C without any additionalstoring conditions. Prior to use exposure of glucose sensors to buffersolution resulted in stabilized signals for micromolar detection insweat.

Fabrication of battery-free NFC-based electronics: A LPKF U4 UV laserpatterned a commercial substrate (Du pont Pyralux AP8535R) to form aflexible printed circuit board (PCB) for the wireless, battery-freeelectronics. Pulsed mode electroplating (LPKFContac S4) filled the viaswith copper to form connections between the top and bottom layers of thedevice. The electronics assembly consisted of soldering themicrocontroller and NFC frontend combination (TI RF430FRL152H), zerocrossover operational amplifier (Analog devices ADA4505-2) and variouspassive resistor and capacitor components in 0201 form-factor, using lowtemperature solder (Indium corp. In/Sn 90/10) paste. Finally, a 14 μmthick layer of parylene formed by chemical vapor deposition (SCSLabcoter® 2 Parylene Deposition System, Specialty Coating Systems, IN)serves as a waterproof encapsulation for the entire system of NFCelectronics.

Working principle of biofuel-cell based electrochemical sensors: Atypical biofuel cell based electrochemical sensor comprises of an enzymefunctionalized anode and an oxygen reducing cathode. The enzymeselectively catalyzes the oxidation of the desired analyte (for e.g.:lactate or glucose) and thus offers selectivity to the biofuel cellbased sensors. In addition to the enzyme, the anode also includes aredox mediator for efficiently shuttle electrons from the enzyme'sactive site to the current collector. The cathode is fabricated bycoating catalysts for oxygen reduction reaction. Oxidases anddehydrogenase enzymes are typically used for selectively oxidizing thedesired analyte. Commonly used redox species, such as, but not limitedto, tetratiafulvalene, quionones, redox dyes act as electron shuttles.Current collectors include, gold, platinum, stainless steel, carbon.Performance of the sensors can be increased by incorporatingnanomaterials such as but not limited to, carbon nanotubes, graphene,metal nanoparticles, metal oxide nanoparticles, etc. The oxygen reducingcathodes include functionalizing current collectors with noble catalysts(platinum black, platinum on carbon, ruthenium on carbon), or enzymessuch as laccase, bilirubin oxidase that reduce dissolved oxygen towater. Both anode and cathode are further coated with polymericmembranes to obviate leaching of chemical reagents, as permselectivelayer to reduce interference from other chemicals and extend thedetection range of the sensor.

When exposed to the sample (sweat), the analyte (e.g. but not limited toglucose, lactate) gets spontaneously oxidized at the anode whiledissolved oxygen gets reduced at the cathode. These spontaneousreactions lead to a flow of current between the two electrodes whosemagnitude is proportional to the concentration of analyte. By applying afixed resistor between the anode and the cathode one can measure theoutput voltage (which is a function of concentration; V=I*R and I αconcentration) using NFC electronics.

Hybrid, battery-free, skin-mounted system for sweat sensing: Theplatform includes two components: a disposable soft, microfluidicnetwork, and a re-usable, thin NFC electronics module. An exploded viewillustration of the overall construction of each of these sub-systems isin FIG. 20A. A low-modulus (˜1 MPa) silicone elastomer, patterned usingsoft lithographic techniques, defines a set of isolated chambers forcolorimetric and electrochemical sensing, a ratcheted channel forquantifying sweat rate and total sweat loss, and a collection ofinterconnecting microchannels with passive, capillary bursting valvesfor routing sweat through the device. A patterned layer ofskin-compatible adhesive enables robust attachment to the skin and itdefines openings as interfaces between the skin and inlet ports in thebottom side of the microfluidic structure. The soft, flexibleconstruction, as illustrated in FIG. 20B, allows comfortable,water-tight, irritation-free mounting onto curved regions of the body.

FIG. 20C shows the electronics module, where the NFC interface supportsboth wireless power delivery and data transmission to any NFC-enabledconsumer device, such as a smart phone, tablet or watch. The designexploits a two-layered flexible circuit with minimal component count anda battery-free configuration for real-time data acquisition from lactateand glucose sensors in a biofuel cell layout located in the microfluidicstructure. The biofuel cell design involves a voltage amplifier withdefined sensor element load implemented with a small footprintoperational amplifier and miniature passive components. The circuitconditions the signal for digitalization within the integrated NFC chip(TI RF430FRL152H). The analog electronics are robust, with minimalsusceptibility to external noise caused by the NFC electronics andfluctuations in the supply voltage.

To enable re-use, the electronics mount onto disposable microfluidicsystems with a releasable electro-mechanical interface. Specifically, aset of thin, small-scale neodymium magnets (diameter, 1 mm; height, 0.5mm) affixed with conductive adhesives to contact pads on the backside ofthe electronic platform and another set embedded in recessed wellsunderneath contact pads to the electrochemical sensors in themicrofluidic platform enable reversible, mechanically robust andself-aligning attachment with low resistance electrical coupling (FIG.20D). FIG. 20E shows a photograph of the complete system. The user firstadheres the microfluidic system to the skin, then magnetically mountsthe electronics on top. An NFC enabled portable device or a long-rangereader placed in proximity initiates wireless, real-time dataacquisition from the lactate and glucose biofuel cell sensors. Visualreadout or analysis of digital images allows colorimetric quantificationof chloride, pH, and sweat rate/loss. FIG. 20F shows a system attachedto the forearm during sweating. In one example of use, the NFCfunctionality in a smartphone enables wireless data extraction and itscamera permits digital colorimetric analysis, as illustrated in FIG.20G.

NFC electronics: FIG. 21A presents a simplified schematic illustrationto highlight that amplification relies on a simple voltage followerdesign with a high frequency filter that eliminates fluctuationsintroduced by the electric field of the primary NFC antenna. This NFCelectronics sub-system magnetically couples to electrochemical sensorsembedded in a disposable microfluidic substrate. FIG. 21B shows acompleted device adhered to surfaces with small radii of curvature todemonstrate the mechanical robustness of this coupling scheme. FIGS.21(C and D) presents the corresponding current (I) vs voltage (V) curvesassociated with the magnetic connection and the variation in phaseresponse for the antenna in response to bending. These results highlightstable antenna performance metrics (i.e. Q-factor and resonance peakposition) even during mechanical deformations and under cyclicalattachment/detachment conditions. FIGS. 21(E and F) displays recordedI-V curves and impedance characterization at frequencies up to 1 MHzcollected at regular intervals during cyclic testing.

Robust operation follows from electrical working principles that aretolerant to fluctuations in supply voltage that can occur during weakNFC coupling to the reader antenna. Because a non-regulated harvestingcircuit scheme yields the highest possible coupling efficiency, theanalog frontend must operate in a manner that is independent of voltagesupply to allow for variances in magnetic resonant power transfer and,thus, stable operation in practical scenarios. This goal is accomplishedby using a zero-crossover operational amplifier that amplifies thesensor signal regardless of supply voltage, without distortion.

Simulation Program with Integrated Circuit Emphasis (SPICE) softwarereveals the behavior of the biofuel cell-based lactate and glucosesensors signal conditioning when subject to varying supply voltage (FIG.25). FIGS. 25(C and D) clearly confirms stable data acquisition over theentire range of supply voltages. FIGS. 21(G and H) show experimentalvalidation through studies of the effect of distance and angle betweenthe device and a hand-held reader with antenna size comparable to asmartphone (5×3 cm2) on sensor signal quality for the case of a constantreference sensor signal (100 mV) applied to the circuit. FIG. 21Gillustrates that the reader records stable signal from the device at adistance up to ˜38 mm. FIG. 21H shows that the reader is capable ofrecording uninterrupted, constant signals from the device at angles upto 600. These results demonstrate the broad range of conditions forwhich reliable data can be acquired, thus highlighting the robust,practical operational capabilities.

Biofuel cell-based electrochemical sensors for lactate and glucose: Thebiofuel cell design for the sensors is a critical feature of thesystems. A scheme that illustrates different components of the lactatesensor is in FIG. 22A, whereby the anode consists of circularly cutcarbon nanotube (CNT) paper that provides a conductive, high surfacearea substrate to immobilize lactate oxidase (LOx) enzyme, forselectively catalyzing lactate oxidation, and the redox mediatortetrathiafulvalene, for shuttling electrons between the enzyme's activesites and the underlying CNT paper. A chitosan and polyvinyl chloridemembrane coat the anode to minimize leaching of the mediator and enzyme,and to extend the linear detection range of the sensor. The cathodeconsists of a functionalized current collector of gold with an overlayerof platinum black, all coated with a Nafion® membrane. The platinumblack acts as a catalyst for oxygen reduction while the Nafion® membraneprevents leaching of platinum black. The fluoride backbone of theNafion® polymer facilitates adsorption of dissolved oxygen onto thecathode's surface, thereby improving the kinetic rate of oxygenreduction. An optical photograph of the complete lactate sensor is inFIG. 22B.

The anodic and cathodic reactions that generate electrical currents inthe lactate sensor are proportional to the concentration of lactate. Aresistor connected across the sensor transforms the current into avoltage-based signal for detection and wireless transmission via the NFCelectronics. The response of the sensor with increasing lactateconcentration, evaluated in phosphate buffer solution at ambientconditions, appears in FIG. 22C. FIG. 22D shows the correspondingcalibration plot, which indicates that the sensor signal stabilizeswithin 300 s and increases linearly with lactate concentration. Thisresponse is reversible (FIG. 22E) as demonstrated in experiments thatinvolve increasing the lactate concentration from 0 mM to 15 mM,reducing it to 0 mM, and then increasing it again in a step-wise fashionback to 15 mM for 4 consecutive cycles. The results highlight linear,reversible responses to time-varying concentrations of lactateconcentrations with minimal hysteresis (FIG. 22H) across aphysiologically relevant range. FIG. 22F displays response at 30° C. inartificial sweat at pH 5.5 while FIG. 22G shows calibration plot forincreasing lactate concentration in artificial sweat with different pHat 30° C.

A similar approach, applied with a few modifications, yields sensors forglucose. Here, glucose oxidase enzyme is directly dispersed in theNafion® to ensure rapid interaction of glucose with the enzyme andconsequent capabilities in detection of micromolar concentrations. Thecathode involves a gold-based current collector coated with a suspensionof platinized carbon in Nafion® solution. FIG. 23A illustrates thedifferent components of the sensor and FIG. 23B presents an image.Comprehensive studies conducted in a manner similar to those for thelactate sensor define the response. FIG. 23C summarizes real-timemeasurements as a function of increasing concentrations of glucose inbuffer at ambient conditions, with a corresponding calibration plot(FIG. 23D). FIG. 23E shows reversible nature of the sensor response.FIGS. 23 F and G illustrate response of sensor in artificial sweat (pH5.5, 30° C.) and effect of pH on sensor response respectively.

Colorimetric assays and microfluidics: The disposable microfluidicsubstrate houses the electrochemical sensors, various colorimetricassays and it supports valves, channels and reservoirs for handlingsmall volumes of sweat delivered into the system by the action of theglands themselves. For chloride concentration, the colorimetric assayrelies on silver chloranilate, a chemical that complexes with chlorideions to generate a species with a distinct purple color. Mixing silverchloranilate with pHEMA solution creates a gel-like suspension thatimmobilizes the insoluble silver byproduct in the assay well. The resultprevents migration of silver particulates during flow of sweat in themicrofluidic channel, thereby eliminating their effects on colorextraction. The extent of change in color determines the concentrationof chloride through a linear calibration curve, as shown in FIG. 24A.This chemical reaction provides a more reliable, accurate colorimetricresponse compared to previously reported alternatives for analysis ofchloride in sweat. Similarly, paper pads coated with a pH sensitive dyeand a phase transfer catalyst serve as a colorimetric means fordetermining pH. The evolution of color as a function of pH over aphysiologically relevant range is in FIG. 24B. Calibration plots revealthe linear relationship between the R value (of the RGB code) atdifferent pH levels. FIG. 26 shows simple color reference bars developedfor each of these calibration plots to facilitate visual or digitalcolor extraction and conversion to concentration.

The part of the system designed to measure sweat rate/loss involves asimple, circular channel with a water-soluble dye located near the inlet(FIG. 24C). The incoming sweat dissolves the dye as it flows past,thereby creating a visible, colored fluid with an easily identifiablefilling front in the channel. The position of this front defines thelocal rate and loss of sweat from the corresponding location on theskin. FIG. 27 shows a linear correlation between data acquired from thesweat rate sensor to the normalized total, or full-body, sweat loss. Inthis particular design, the channel volume (˜58 μl) and the dimensionsof the inlet interface to the skin allow tracking of sweat loss for upto 6 hours based on an average sweat rate in the range of 12 to 120ml/hr/cm².

Although these assays have an irreversible response, time dependentchanges in sweat composition can be captured by using fluidic designsthat enable time-sequential sampling (chrono-sampling) of sweat usingpassive valve constructs. The example in FIG. 28A uses collections ofcapillary bursting valves (CBVs) to enable sequential filling of aseries of independent microreservoirs, each pre-impregnated with acolorimetric reagent. The left and right sides of the device providechrono-sampling analysis of pH and chloride, respectively.

Since the electrochemical sensors for lactate and glucose arereversible, a single-chamber design with a single channel to divertsweat from this chamber to an outlet is sufficient. These twomicrofluidic structures flank either side of the patch. The sweat sensorchannel resides in between the regions for electrochemical andcolorimetric sensing. FIG. 28B and FIG. 24D highlight the multimodalmicrofluidic substrate and chrono-sampling features of the system.Circular holes (diameter, 1 mm) serve as inlets in the base of themicrofluidic platform for the sweat rate, chloride and pH sensors whileellipsoidal shaped holes (major axis, 5 mm; minor axis, 3 mm) act asinlets for glucose and lactate sensors. The skin adhesive layer hascorresponding circular (diameter of 3 mm) and ellipsoidal (major axis, 6mm; minor axis, 4 mm) openings.

TABLE 1 Comparison of data acquired from sensor patch and conventionaltechniques during human trials. Hybrid Conventional Subject ParameterSensor Patch technique Subject #1 Chloride 62 ± 5 mM 66 mM 36 ± 5 mM 43mM pH 6.3 ± 0.05 6 6.2 ± 0.03 5.5 Lactate 10.4 ± 0.1 mM 17.5 mM Glucose23.2 ± 2.4 μM 53 μM Subject #2 Chloride 34 ± 2 mM 40 mM pH 6.4 ± 0.1 6.5Lactate 19.3 ± 0.5 mM 28 mM Glucose 52 ± 14 μM 100 μM

Example 6: Resettable Epidermal Microfluidic Sweat Loss Sensor (U.S.Pat. App. 62/514,520)

Current methods for measuring sweat volume loss from the skin rely onabsorbent pads taped to the skin, but do not offer the ease of use insweat capture needed for quantitative or real time tracking overmultiple uses. Described herein is a thin, soft, “skin-like”microfluidic platform is introduced that bonds to the skin to allow forcollection and storage of sweat in an interconnected set ofmicroreservoirs. A visual indicator of sweat volume is formed byexploiting refractive index differences between air, sweat, and thedevice layers.

Provided herein is a resettable, real-time sweat loss monitoringmicrofluidic device. The device allows for resetting or draining of thereservoir chambers during use, allowing for multiple monitoring periodswithout requiring a new device. An optically based water indicator maybe provided into the microfluidic flexible substrate, allowing for thedetection or monitoring of biofluids without single use indicators suchas water indicator tape or CoCl₂.

Two methods of indicating when sweat is present in a microfluidicchamber are provided, both of which exploit differences in refractiveindex. FIG. 29 illustrates a nano/micro patterned grating in ahydrophilic polymer which diffuses light when the chamber is empty, buttransmits light when the chamber is full. A micropatterned grating in ahydrophilic surface scatters light when there is a refractive indexmismatch and transmits light when the mismatch is negligible. When lightis able to travel past the grating, the colored indicator is visible.n=refractive index.

FIG. 30 illustrates a similar concept, but utilizes a hydrophobicpolymer surface and a nano/micro patterned grating to trap air bubbleswhich reflect light when the chamber is full. Patterned features in ahydrophobic surface trap air bubbles when filled with sweat and reflectincident light, changing the appearance of the colored indicator.

FIG. 31 illustrates a potential method of resetting the sensor to itsinitial state. Dual expunge ports reduce the likelihood of accidentalsweat discharge, but when covered and pressed simultaneously empty thechamber and reset the device back to its initial state.

Example 7: Microfluidic Systems for Epidermal Sampling, Sensing andTissue Actuation

Sweat induction through hot shower/bath: Sweat induction has beenreported through daily (heavy) exercise, sauna, and iontophoresis.However, daily (heavy) exercise limits the application of epidermalsweat sensing for disease diagnostics and health monitoring for patientswho often present multiple comorbidities. The high cost andinconvenience of sauna prevent its wide use for sweat stimulation. Theiontophoresis process involves the delivery of stimulating agents to thedesired sweat glands with the aid of electrical current. This processinvolves complex electronics for power delivery. In addition, theelectrode corrosion and burning can cause discomfort in subjects. Herewe report a patient-friendly, low-cost, and convenient method usinghot-water shower/bath for sweat induction. A soft, epidermalmicrofluidic device that captures and routes sweat through a network ofmicrochannels and reservoirs adheres and conforms to skin. The followingprocedures will be involved to extract sweat through hot-watershower/bath. First, take a hot shower/bath, which usually takes 15-30minutes to induce sweat, with water temperature around 43° C. Dry thebody with a clean towel. The application sites are prepped with adisposable alcohol prep wipe (Dyanrex) to remove skin oils and dirt(FIG. 33a ). Second, sweat patch is placed on up to five locations onthe body including upper arm, chest, lower back, upper back, andforehead (FIG. 33b ). Third, stay in the shower room to keep sweatinguntil one reservoir in each part is filled. It usually takes 15 to 30minutes. Devices are left on for less than one hour. Finally, takephotographs of each device using a cell phone camera. Photographs areused for data analysis. Epidermal microfluidic device is removed bygently pulling the adhesive and sensor off the subject. Skin surfacesunderneath are cleaned immediately with an alcohol wipe, and devices aredisposed of and replaced after each use. FIG. 33c shows representativeoptical images of microfluidic device spotted with blue dye that mixeswith sweat using hot-water shower/bath. FIG. 33d shows the number ofreservoirs filled with sweat from the inlets of 1-3 on forehead, chest,axilla, and back. This study suggests that hot-water shower/bath caninduce large volume sweat for on-demand and in situ analysis. It opensthe door for the application of sweating sampling and sensing in diseasediagnostics and health monitoring.

Example 8: Thin, Soft, Skin-Mounted Microfluidic Networks for Detectionand Analysis of Targets of Interest in Sweat

Described herein is a thin, soft, “skin-like” microfluidic platform isintroduced that bonds to the skin to allow for collection and storage ofsweat in an interconnected set of microreservoirs for the quantitativeanalysis of different targets of interest. Quantitative analysis caneither be performed on the device or after collection via elution ofsweat for external lab analysis. This platform is suitable for an arrayof applications including disease diagnostics through quantitativeanalysis of sweat chloride concentration for cystic fibrosis screening,monitoring of kidney health by measuring urea content in sweat, clinicaland personal alcohol screening to quantify alcohol consumption, drugdetection/screening, and personal/clinical glucose monitoring bothcontinuously and at periodic time intervals. Each use case harnesses thesoft, flexible mechanics, integrated sensors, and microfluidic handlingof sweat to achieve precise, accurate, and quantitative measurementssuitable for both clinical and personal health monitoring.

The provided systems and methods are useful for collecting andrecovering biofluid such as sweat, blood from the epidermis for diseasediagnosis, for example, by analyzing biomarkers in the biofluid.Additionally, the systems and methods are useful in collecting andanalyzing organic and inorganic chemicals in sweat for home monitoringand self-quantification of conditions (e.g. drug screening, alcoholcontent monitoring).

Provided is a single device for collecting sweat and analyzingbiomarkers or other targets of interest. A self-adhesive is used tostick the device to the subject, so no additional skin-attachmentassistance such as tourniquets or gauze. The device has a conformal,skin-compatible design for storage and final extraction of sweat. Thedevice utilizes microfluidics to allow for analysis of small volumes ofsweat. Further, the device may have a wireless connection to analyticalcomponents (smartphone, computer)

Described are systems and methods for collection and storage of a liquidin a microfluidic channel network for either in-situ or external,lab-based analysis. The device is in a soft, flexible configuration thatenables conformal attachment to the epidermis promoting sweat collectionwhile preventing loss. The device allows for the collection of eitherlarge or small volumes of sweat and performance on-board analysisthereby enabling custom-tailored, rapid disease diagnosis and/orscreening.

The described device is suitable for screening a wide array of targetsof interest including biomarker concentration, such as chloride, forcystic fibrosis screening, organic/inorganic compounds for monitoringalcohol or drug consumption (such as marijuana), monitoring dialysisefficacy for patients with kidney failure (urea content in sweat),continuous (or discontinuous) monitoring of glucose levels in sweat, andother clinically/health relevant markers for disease screening,monitoring, and diagnosis.

The thin construction and soft mechanics of this device enablesconformal attachment to the skin for the purpose of collecting, storing,and analyzing sweat. Overall, the device geometry may be both circularand rectangular in form. Radial geometries enable sweat extraction viacentrifugation after the device is removed from the skin. Rectangularand radial geometries enable sweat extraction via pipetting or via apurpose-built extraction tool.

Advantageously, devices may be optimized for biofluid extraction. FIG.34 illustrates microfluidic networks optimized to be filled and to storebiofluids in discrete amounts without the trapping of air so that youcan fully extract the biofluid with approximately 100% efficiency.Clinically it is advantageous to have a discrete volume of fluid, but,in some embodiments, a continuous channel for sweat storage with avisual design that provides the simulation of a “reservoir” to the humaneye is implemented. The design is similar to a pipe designed to looklike a bowl. While a bowl provides more visually apparent volume to aviewer, it may also trap air during filling or trap fluid duringextraction. In contrast, in a pipe fluid flows easily but doesn'tprovide as clear visual volume information. The design utilizes bothbenefits by combining a pipe into a bowl shape for facile fluid fillingor extraction without air entrapment while providing clear visual volumeinformation. The benefit of this design is illustrated in FIG. 35. Thechannels are designed so that they offer information about the currentstate of collection. Fluid changes direction based upon a particularfilling condition (¼ full, ½ full, of a target volume) providing havereal-time, visual information about the current state of collection.Thus, the overall image of the device dynamically conveys information(e.g. visually).

Devices are fabricated from layers of poly(dimethylsioxane) (PDMS) orother soft polymer supported upon either medical-grade acrylic adhesivefilm or on a custom spin-on adhesive composite for bonding to the skin.Materials used in device fabrication can be tailored for specificapplications such as attachment to newborns, collection of volatilecompounds, or long term drug monitoring (such as providing evidence oftampering with device). An exemplary multilayer device is provided inFIG. 36. Further, in some devices, the device is epifluidic (e.g.controls the expose skin surface area to the device). This allows theamount of sweat and the collection rate into the device to be modifiedor “tuned” by creating prescribed patterns in the adhesive or theskin/device interface layer. Patterning allows optimized collection withmaximum adhesion (see FIG. 36).

By controlling the skin surface area and thus collection area of thedevice, the efficiency and performance of the device is increased ascompared to other collection methods (FIGS. 35 and 36). The same volumeof sweat is collected in the same time, but with much less area exposed.This is due to the soft mechanics of the device, including flexible,conformal, and air/water tight sealed against the skin devices.

Devices are designed to extract specific volumes of sweat in a specifiedtimeframe with particular embodiments fabricated with different fluidicchannel widths, heights, and layouts. For a representative device, thefirst layer defines the microfluidic channel network (total thickness400 um). The second layer has no molded features and serves as thecapping layer (thickness 200 um) to the first layer to form enclosedchannels. Both layers are fabricated from PDMS. The third layer formsthe adhesive bond to the skin. Fluid (sweat) is routed into the devicefrom the skin by way of an inlet formed by removal of the PDMS cappinglayer/adhesive layer. The diameter of the inlet determines how manysweat pores are sampled by the device.

A sweat chloride test is the gold-standard diagnostic method used forscreening cystic fibrosis whereby the quantitative analysis of the sweatchloride concentration is measured in microliter volumes of sweatcaptured from targeted collection sites on the skin by externallaboratory methods. Current sweat test collection methods rely on eitherabsorbent pads taped to the skin or a commercially available product;however, both methods are limited by ease of use, sample contamination,and poor sealing with the skin, especially when used for neonatal cysticfibrosis screening. A thin, soft, “skin-like” microfluidic platform thatbonds to the skin to is advantageous as it allows for collection andstorage of sweat in an interconnected set of microreservoirs.Quantitative analysis can either be performed on the device or aftercollection via elution of sweat for external lab analysis.

A sweat sample captured via a sweat test is analyzed in a laboratorywhere coulometry is used to determine the concentration of chloride.While the foundational operation of the epifluidic device is thecollection of sweat in a sweat test, quantitative chloride analysis canalso be performed through integrated electrical sensors or viacolorimetric analysis. By integrating ion-selective electrodes forchloride into the microfluidic network, electrochemical analysis of thechloride concentration can be performed in real-time as the sweat entersthe device during elution. Using near-field communications, this datatransfer and sensing can be conducted wirelessly. Furthermore, throughcontinuous monitoring of the concentration, sweat rate can be measuredand validated against the minimum sweat rate necessary for a validcystic fibrosis test. As a result of the device flexibility,colorimetric analysis can also be performed either separately orsimultaneously with electrochemical analysis to determine chlorideconcentration using commercially available colorimetric chemical assays.

Devices for either acute or chronic drug screening are fabricated eitherfor use in a clinical environment or as a temporary, at-home monitoringfor analysis either by the wearer or by a clinician. Devices must betamperproof if worn for home monitoring achieved via means such asdestruction of sensitive components in the device itself (embedded art,fragile device construction in shear, broken indicator areas), stainingof the skin with a colored dye, or electronic recording stored on thedevice so as to protect the integrity of the measurement. The samerequirements are necessary for the clinic, but with a decreased need forlong-term (˜24 h) stability as tests are typically shorter (˜10 min).Screening itself is achieved either via collection of sweat for externalanalysis, integrated electronic detection of key markers of drug/alcoholactivity, via colorimetric analysis of key markers, or a combinationthereof. Individual device barcoding will be required for drugscreenings necessitated by legal requirements/chain of custody.

Devices for personal alcohol testing are fabricated in similar manner toother devices. Designed for personal use, these devices integrategraphics to provide clear, simple read-outs to accurately gauge alcoholconsumption versus time. As aesthetics are important for use in anon-clinical setting the devices are designed to have as minimal afootprint as possible. Sensing is performed using either colorimetric orintegrated electrode approaches. Key points of distinction are formfactor, sampling volume, and chrono-sampling. Discrete monitoring isalso possible via wireless sensing coupled with a smartphone.

Devices for glucose monitoring are fabricated in similar manner to otherdevices. Designed for both clinical and personal use, these devicesintegrate graphics to provide clear, simple read-outs to accuratelygauge glucose over a specified time. Sensing is performed using eithercolorimetric or integrated electrode approaches depending uponmeasurement requirements. Key points of distinction are form factor,sampling volume, and chrono-sampling. Discrete monitoring is alsopossible via wireless sensing coupled with a smartphone.

Multiple stacked microfluidic network device layers providemultifunctionality to the epifluidic sweat collection and analysisplatform. Benefits include increased sweat storage in same epidermalsurface area, independent collection areas for on-device controls,inclusion of active components (valving, electronic sensing), multipleanalytical channels (electronic, colorimetric, external lab), andincreased collection rates via multiple inlets. Multiple microfluidicnetwork layers can be interwoven with graphical constructs to provideadditional functionality by interacting dynamically with printed images.

Integration of microlenses (e.g. cylindrical, hemi-spherical) into themicrofluidic channel network can improve the accuracy of thecolorimetric assay performance by either increasing the effectpathlength for light to pass through the device or by collecting morelight scattered by the regions of interest (e.g. microfluidic channels).Additionally, integration of lenses into the device offer increasedcomplexity for integration of art into the device such that the art caninteract dynamically with the measurement in real-time.

To provide sensing capabilities, active components such asphotodetectors, laser diodes, vertical cavity surface-emitting lasers(VCSEL), waveguides, optical resonance cavities can be integrated intothe device. Additionally, device surfaces or composition can be modifiedto provide additional sensing capabilities through integration ofplasmonic nanoparticles (e.g. gold nanorods) that respond to thepresence of different analytes of interest. These components provideenhanced sensitivity to the described sensing requirements.

Molds for fabricating these devices can be produced using standardcleanroom processing techniques, via refined additive manufacturingprocesses, or via micromilling. A molding process is used to produceaccurate (˜50 um channel width) channels using 3D printing via selectivelaser sintering of high-temperature photocurable resin. Whereas otherphotocurable resins warp at the temperatures required to cure polymerssuch as PDMS, the combination of photocurable resin with 3D printingenables production of physically stable molds at the temperaturesrequired for fabricating these devices. Mold production via micromillingaluminum also provides a method to rapid prototype molds with extremelyfine resolution (˜100 um channel width, >30 um depth).

A composite material comprised of soft-skin adhesive (e.g. Dow Corning)with uncured PDMS precursor is used to fabricate layers of soft polymersuitable for reversible bonding to virgin PDMS with a bond strengthsufficient for a fluid-tight seal. Beneficially, neither heat nor oxygenplasma are required to form sealed, flexible, soft epifluidic devices.Furthermore, the reversible nature of the bond enables reusable devicefixtures (e.g. electronics) with disposable fluidic networks.Formulations of different composite mixtures (30:1, 40:1, 50:1) providedifferent adhesive strength suitable for a variety of applicationsincluding bonding to electronic components and temporarily sealingchannels for surface treatment/activation. Absence of surface plasmaactivation or heat treatment enables integration of sensitive assays(enzymes) or rapid prototyping while maintaining same surface chemistry.

We have demonstrated an analytical platform for the diagnosis of cysticfibrosis that exploits ultrasoft, conformal, “skin-like” microfluidicchannels to collect sweat from eccrine sweat glands stimulated viapilocarpine iontophoresis. A representative device, shown in FIG. 37A,has an overall circular geometry with a diameter of 34 mm. The radialconstruction allows for both maximal sweat collection of a stimulatedregion (30 mm, diameter of Wescor Pilodisc) and attachment to multiplebody locations (e.g. forearm, thigh) on multiple subjects (e.g. infants,adults).

The device, comprised of three layers of soft, medical-grade siliconeelastomer (polydimethylsiloxane, PDMS, Dow Corning) exploits thingeometries and soft mechanics to enable intimate, conformal bonding tofragile newborn skin. This bond is a zero-pressure, fluid-tightinterface between the device and the skin formed via a medical-grade,irritation-free, FDA-approved gentle skin (i.e. newborn safe) adhesive(3M silicone adhesive; thickness, 100 μm). Laser-patterned openingsdefine the sweat harvesting regions through which sweat, driven by thesweat gland pressure (˜3 kPa) passes into one of three independentchambers (FIG. 37B), which each store up to 70 μL of sweat [11].Optimization of the adhesive pattern maximizes sweat collection (70 mm²exposed surface area per region corresponding to ˜100 sweat glands)while maintaining conformal adhesive contact during a sweat test.

The device comprises an embedded microfluidic network of embossedchannels (500 μm width, 350 μm uniform depth) in a soft (˜145 kPa) PDMSlayer (thickness, 400 μm) [10]. A capping layer (thickness, 100 μm)serves to seal the first collection layer in which a single, continuouschannel comprises a collection chamber with an inlet (open to skin) andan outlet to the second collection layer. This second layer containsembossed channels (500 μm width, 300 μm uniform depth) which form threesecondary collection chambers connected to the first layer chambers byindependent inlets. The middle capping layer also serves to seal thesecond layer microfluidic channel network. The first layer chambers eachhold 50 μL of sweat while the second layer chambers each hold anadditional 20 μL of sweat. Constructed in a tiered manner (first layerdiameter, 34 mm; second layer diameter, 20 mm), the variable thicknessof the device (edge thickness, 500 μm; center thickness, 900 μm),coupled with the soft material properties of the PDMS, improves deviceflexibility (FIG. 38). The thin, soft, compliant device constructionprovides a key differentiating factor between this device and thecurrent FDA-approved technology (e.g. Macroduct® sweat collectionsystem) as it enhances the conformal coupling to the skin, especiallyfor neonates.

Optimization of the microfluidic channel design enables maximized sweatcollection after pilocarpine iontophoresis. FIG. 39 shows a sequence ofoptical images of the device immediately after sweat stimulation, 15 mininto the collection, and at the conclusion of a sweat test (30 min). Thechannels on the first layer contain reservoirs of teal dye (FDAapproved, verified chloride-free) to visualize the flow of sweat as soonas sweat enters the device. As the device samples from three independentregions within the stimulation area, slight variations may result due tothe biological variation of sweat gland density. The use ofmedical-grade soft skin adhesive provides a robust, water-tight bondbetween the device and skin promoting the complete and rapid collectionof sweat without the application of a tourniquet. This eliminates asignificant risk to neonates, especially when the diameter of the arm issmaller than the size of a Macroduct® sweat collection system device. Asthe adhesive layer provides an optimal balance between bond strength andsweat collection (evaluated by the absence of leakage), rather thanhigh-pressure contact, the collection device does not suffer frommotion-induced collection failures.

An additional design consideration of the collection device is theefficacy of sweat extraction and ease-of-extraction. Not only must sweatbe fully extracted from the device, but the extraction mechanics mustpromote ease of operation and eliminate potential sources of chloridecontamination. The location of the collection inlets (i.e. opening toskin) in the central region of the device (FIG. 37) eliminates sweatleakage upon the removal of the device from the skin as the mechanics ofremoval (i.e. vertical lifting from the device edge) creates a temporarypumping action to transport sweat contained in the channel farther intothe collection chamber as observed in positional difference between theoptical image in FIG. 39 (30 min) and that of FIG. 40A. The onlyadditional equipment required to extract sweat from the collectiondevice is a standard pipette (1 mL, generic). The device-layer inletsize (1.2 mm diameter) is smaller than the pipette opening (1.5±0.1 mmdiameter, brand dependent) so that when the pipette contacts theelastomeric device a strong, temporary water-tight and gas-tight sealforms so that a negative pressure occurs in the microfluidic channelupon extraction with the pipette. This negative pressure is sufficientto fully extract sweat from each chamber, regardless of filled volume.The sweat extraction rate is linearly proportional to the amount ofnegative pressure applied (set volume on a variable pipetter) and rateof draw on the pipetter. The combination of extracted sweat from allthree collection chambers defines the total collected sweat volume.

Example 9: Multilayer Channel Construction

For certain applications, the device area must be restricted to certainregions of skin or must undergo aggressive bending to conform to a highradius of curvature (e.g. arm of an infant). For these cases, the devicegeometry can be structured to harness the device design in the verticaldimension. FIG. 41 is a render of a representative device stack thatincorporates multiple levels to provide expanded functionality to asweat device not possible in a planar geometry. FIG. 41A is a cappinglayer providing fluidic access to the layer in FIG. 41C and anair-escape access to the layer in FIG. 41B. FIG. 41B is a layer for anintegrated colorimetric assay that provides independent analysis of thefluid collected in the layer FIG. 41C. FIG. 41C is a collection channelnetwork that collects fluid for external analysis (extracted via thelayer in FIG. 41A). The layer in FIG. 41D is the skin-interface layerthat collects sweat from the skin and directs it to the independentchannels in FIGS. 41B and 41C. FIG. 42 shows a representative deviceusing a multistack layer construction.

Example 10: Resettable Epidermal Microfluidic Sweat Loss Sensor

The black indicator layer is formed by spin coating 10:1 PDMS containing1.5 wt % black pigment and 1.5% white pigment on a flat PMMA coatedwafer. The clear patterned layer is formed by spin coating 10:1 PDMS ona PMMA coated silicon wafer with bas-relief features. Both layers arecured at 100 C for 1 hr. The scattering material is a commerciallyavailable hydrochromic ink (LCR Hallcrest H151000). The exactcomposition is unknown. The hydrochromic ink is dispersed in water (5:1wt water:ink) deposited via air brushing onto the molded PDMS layer anddried at 100 C for 5 min. Scotch tape is used to remove the ink that isdeposited outside the microchannel. Corona treatment of the molded andflat PDMS layers prepares the layers to be bonded. Lamination, lightpressing, and heating at 70° C. for 24 hrs ensures a permanent bondbetween the layers and completes fabrication.

Example 11: Thin, Soft, Skin-Mounted Microfluidic Networks withCapillary Bursting Valves for Chrono-Sampling of Sweat and MeasuringPressure from Sweat Gland and Colorimetric Detection of Chloride

Soft, multi-functional microfluidic device for colorimetric sweatanalysis: A soft microfluidic device made from PDMS has flexibility andinterfaces to the skin (FIG. 1a ). The device provides severalfunctionalities that: 1) analyzes of concentration of chloride, glucose,pH and lactate in sweat, 2) temperature of sweat by colorimetric method,3) calculates local sweat loss and instantaneous sweat rate via adhesivelayer that provides a water-tight sealing between skin and the devicethat enables the device to collect sweat continuously (FIG. 1b ). Sweatgland under open region of skin under adhesive generates sweat flowabout 2 kPa to 1) inlet #1 and fill the serpentine channel whiledeveloping color to detect chloride concentration in sweat and shows thelocal sweat loss, 2) inlet #2 and fills the collection chambers inclock-wise sequential manner through the guide of series of capillarybursting valves and develop color for detection temperature, glucose, pHand lactate of sweat. For colorimetric analysis, each chamber hasthermochromic liquid crystal sensor or chemical assays that developcolor according to the temperature or target biomarker in sweat andcolor reference markers placed around the chamber provide a standardcolor of target temperature or concentration for accurate color analysisthat is not affected by light condition. The exploded view of the deviceshows the detailed compositions of one device (FIG. 1c ). The adhesivelayer attaches the PDMS device on to the skin and the hole in theadhesive opens a route for the sweat from the region to enter to themicrofluidic channels. White microfluidic PDMS channel layer formed bysoft lithography has two channels: the left serpentine channel formeasuring chloride concentration and sweat rate, the right sequentialcircular chambers for measuring glucose, pH and lactate concentration insweat. The depth of the channel is 600 μm and its relatively thick depthprovides sufficient color difference between concentration from thechamber for accurate detection of biomarker of sweat using colorimetricmethod. The chemical assay components are located in each chambers andchannel for their purpose. A 200 μm thick clear 10:1 PDMS capping layercoated with stick PDMS from fully cured 50:1 PDMS generates the closedchannel to the microfluidic layer. The sticky PDMS adhesion is preferreddue to it does not require any heating process or oxygen plasmatreatment that could affect to the stability of chemical assay in thechambers. On top the capping layer, a 25 μm thick thin PET film withreference color marker provide an accurate color analysis. FIG. 1d showsthe process of 1) collecting sweat from exercising, 2) taking picture bysmartphone camera, and 3) analyzing color from the chambers to calculatethe sweat concentration. Comparing the color value from the reactionchamber with values from color reference marker estimates the sweatconcentration in chamber.

Device Fabrication: Fabrication begins with making a silicon wafer mold.Patterning photo-resist of KMPR 1010 (Microchem, MA, USA) on 1 mm thickSi-wafer and deep reactive ion etching (STS Pegasus ICP-DRIE; SPTSTechnologies, Newport, United Kingdom) generated a mold for microfluidicchannel. Thin layer of poly(methylmethacrylate) (PMMA; Microchem, MA,USA) formed on the mold. Pouring 10:1 PDMS (Sylgard 184; Dow corning,MI, USA) mixed with white silicone dye (Reynolds Advanced Materials) at10% wt on the mold and spin coating at 150 rpm with baking at 150° C.for 3 min yielded a 700 μm thick layer. All the chemical assays werelocated on the cured PDMS channel. Sequential process of pouring 10:1and 50:1 PDMS and spin coating at 400 and 1000 rpm baking at 150° C. for3 min yielded a 200 μm thick layer and 75 μm thick layer, respectively.50:1 PDMS provided a sticky layer to bond between microfluidic channellayer and capping layer. 25 μm thick clear polyester film (THERMLfilmSELECT® 10852; FLEXcon, MA, USA) on the top of the device with colorreference marker. 60 μm thick medical grade acrylate adhesive (1524; 3M,MN, USA) bonded to the bottom of the device through 30 sec of laboratorycorona treater (Electro-Technic Products).

Color development and reference marker: Colorimetric method fordetection of biomarkers needs a color reference marker for accurateanalysis of color regardless of light condition. FIG. 45a shows thecollection of color reference markers for analyzing temperature,chloride, glucose, pH and lactate from sweat. For the preparation of thecolor reference marker, in vitro test with standard solution producedreference color and digital imaging and image analysis provides colorvalue of each assay. From the values, the color reference marker isgenerated and printed on the thin and clear film and attached to the topof the device. Mixture three kinds of thermochromic liquid crystals, 40wt % cholesteryl oleyl carbonate (COC), 40 wt % cholesteryl nonanoate(CN), and 20 wt % cholesteryl 2, 4-dichlorobenzoate (CD) encapsulated bythin PET film with black background provides temperature sensor that hasred-start at 32° C., green-start at 33° C. and blue-start at 34° C.enables to detect the temperature from 31° C. to 37° C. (FIGS. 45b ,46). Silver chloranilate immobilized in pHEMA produces purple coloredion from reaction with chloride ion sweat and the color levelcontinuously decreases with chloride concentration that allow lightness(L) level to provide a representative number of the color of assay (FIG.45c ). As the sweat continuously flow the chamber, the color developmenthas a chance to be sensitive to flow rate. Sufficient reaction time fromlong reaction area provide uniform color development independent to flowrate from 1 to 5 μl min⁻¹ (FIG. 47). Glucose in the sweat produceshydrogen peroxide (H₂O₂) from the enzymatic reaction with glucoseoxidase and peroxidase reacts with glucose substrate dye using H₂O₂results yellowish color that changes blue level in the chamberdominantly (FIG. 45d ). Universal pH dye provides a pH sensor and redlevel from the sensor that changes dominantly with pH of solution servea comparing parameter of the color of assay (FIG. 45e ). Lactate assayfollows the similar enzymatic reaction as glucose assay and produces redcolor at low concentration at 5 mM and yellow color from 15 mM. Thegreen level changes dominantly and serves a representative color valuefrom the assay (FIG. 45f ). For enhancement of the color differencebetween concentration that makes more accurate color detection, thechannel depth makes a dominant effect because thicker chamber produceslonger path length of light (FIG. 48). As the chamber thickness definesthe total thickness of the device and volume of chamber, 600 um thickchamber provides soft mechanics of device and appropriate volume ofsweat into the device as ˜6 μl.

Colorimetric assay: 1) chloride: 8 μl of mixture of 50 mg of silverchloranilate (MP Biomedicals, CA, USA) and 200 μl of 2% pHEMA providesassay for chloride detection.

2) glucose: 1.0 μl of buffer, 0.5 μl of substrate, 0.5 μl of enzymelocated in a chamber developed color for glucose detection. (GlucoseColorimetric Assay Kit II; Biovision, CA, USA)

3) pH: pH cocktail solution was realized by thoroughly vortexing 4 ml ofuniversal pH dye (Fisher Scientific, NH, USA), 274 mg of polyvinylchloride (M.W. 233,000, Sigma-Aldrich, MO, USA), 635 μl ofo-nitrophenyloctylether (Sigma-Aldrich, MO, USA) and 508 μl of aliquotin 10 ml of tetrahydrofuran (Sigma-Aldrich, MO, USA) till a homogenoussuspension was obtained. Thereafter, a filter paper was dipped in thecocktail solution for 10 s and allowed to dry at ambient conditions for15 min to realize the solid-state pH assay. Finally, a metal punch(diameter, 2 mm) was used to excise circular pads of the pH assay paperfor incorporating in the wearable patch.

4) lactate: the lactate assay cocktail was prepared by thoroughly mixing17% v/v dye, 17% v/v peroxidase from horseradish (HRP) (20 mg/ml;Sigma-Aldrich, MO, USA) and 66% v/v lactate oxidase (LOx) (60 mg/ml,activity of 101 U/mg; Toyobo Corp., Osaka, Japan) solution. The dyesolution was earlier prepared by mixing 0.5 M3,5-dichloro-2-hydroxy-benzenesulfonic acid (Sigma-Aldrich, MO, USA)with 0.25 M 4-aminoantipyrine in 1:1 v/v ratio, while the enzyme and dyesolutions were prepared in 0.1 M sodium phosphate buffer (pH 7.0) anddeionized water respectively. The lactate assay spot was prepared byfirst coating 2 μl of lactate assay cocktail in the designated chamberof the patch and letting it dry. A second coat of 1.5 μl of enzymesolution containing HRP (20 mg/ml) and LOx (60 mg/ml) in 1:2 v/v ratiowas applied to the assay spot to extend the detection range up to thephysiologically relevant lactate concentration and to enhance the colorcontrast. The assay spot was allowed to dry for at 1 hour at ambientroom temperature before utilizing if for lactate detection.

Colorimetric Temperature Sensor:

A thermochromic liquid crustal is fully sterol-based ternary mixturecontaining 20 wt % cholesteryl oleyl carbonate (COC, Sigma-Aldrich, MO,USA), 40 wt % cholesteryl nonanoate (CN, Sigma-Aldrich, MO, USA), and 20wt % cholesteryl 2, 4-dichlorobenzoate (CD, Pressure Chemical Company,PA, USA). The mixture was heated at 200 C with magnetic stirrer untilforming a homogeneous mixture and was applied on the PET film withprinting black for background and covered by another PET film. A CO₂laser (Universal Laser Systems, AZ, USA) defined the size of the TLCfilm as 2.5 mm in diameter.

Standard Color Development and Color Reference Marker Preparation:

Sodium chloride, D(+) Glucose and L(+) lactic acid (Sigma-Aldrich, MO,USA) generated standard solutions in DI water as its concentrations. pHbuffer solution was made and pH meter (Mettler Toledo, Greifensee,Switzerland) measured it. A syringe pump (Harvard Apparatus, MA, USA)generated flow at 1 μl/min speed into the microfluidic device withchloride assay on the hot plate at 31° C. until the solution filled 20%of the channel. For glucose, lactate and pH test, pipetting flowedstandard solution into the chambers. For full color development, thedevice with glucose and lactate assay filled by the solution stayed onthe hot plate at 31° C. for 20 min and pH for 5 min. A digital SLRcamera (EOS 6D; Canon, Tokyo, Japan) took the picture of the device.Photoshop (Adobe Systems, CA, USA) provided color extraction from thecolor in the chambers. A color laser printer (C454 PS; Konica Minolta,Tokyo, Japan) produced a reference maker on PET film at 1200 DPIresolution. The printed the reference marker placed on the device againand smartphone camera (Iphone 5s; Apple, CA, USA) took picture of thechamber with reference marker. The color analysis compared the colorlevel from the chamber and reference marker. Three spots from eachchamber and reference marker provided the average color value. Byadjusting brightness of the image, repetition of printing and comparingprovided the optimum reference marker. For in vitro accuracy test, thecolor developed device with reference marker placed in laboratory withwhite light bulb and yellow light bulb and in outdoor.

Accuracy Test of Colorimetric Methods in Various Lighting Condition

The absolute color value from the image of assay chamber changesaccording to the lighting condition. The color reference marker attachedto the device around the assay chamber represents color value ofspecific concentration and changes its color according to the lightingcondition; this provide accurate color evaluation regardless of lightingcondition. For validation of the functionality and accuracy ofcolorimetric method coupled with color reference marker, the devicessupplied with known standard concentration produces images in whitelight bulb, yellow light bulb and daylight condition (FIG. 49a ).Overall the accuracy of chloride, glucose, pH and lactate are about 5%,10%, 2%, 10% of testing concentration, respectively (Table 2).

The type of lighting condition does not affect accuracy in general. Incase of pH and lactate, daylight condition produces lower estimatedconcentration than expected concentration (Table 2).

TABLE 2 Accuracy of colorimetric method. concentrations (mM) Chloride 2550 75 100 White bulb 23 48 78 101 S.D. 2 1 4 3 Yellow bulb 23 50 76 97S.D. 3 1 1 1 Daylight 25 50 80 102 S.D. 1 0 3 5 Concentrations (μM)Glucose 25 50 75 100 White bulb 20 53 82 101 S.D. 4 4 2 9 Yellow bulb 2352 80 106 S.D. 6 2 3 5 Daylight 21 52 73 104 S.D. 5 1 2 5 pH pH 5.0 5.56.0 6.5 White bulb 4.9 5.5 6.0 6.5 S.D. 0.1 0.0 0.0 0.1 Yellow bulb 5.05.6 6.1 6.6 S.D. 0.1 0.0 0.1 0.1 Daylight 4.9 5.6 6.0 6.5 S.D. 0.1 0.00.1 0.1 Concentration (mM) Lactate 5 10 15 20 White bulb 5.7 10.4 14.819.3 S.D. 0.4 0.4 0.3 1.1 Yellow bulb 5.9 10.3 14.9 20.1 S.D. 0.3 0.51.2 2.2 Daylight 4.6 9.4 13.4 18.4 S.D. 0.8 0.5 0.2 1.4

Example 12: A “Skin-Like” Wearable Microfluidic Sensor for FluorometricSweat Analysis

Layer structured microfluidic system for fluorometric assays: Afluorometric sweat-sensing system consisting of a wearable microfluidicdevice and a smartphone-based fluorescence-imaging device to analyzebiomarkers in sweat in-situ with a simple procedure and highsensitivity. Microfluidic device composed of a multilayer stack of threesubsystems: an adhesive membrane, a sealed microfluidic channel andreservoirs, and a detachable black light-shielding film provides areaction chamber to analyze various biomarkers by fluorometric methods.The micro-patterns in the fluidic layer enable use of fluorometricassays and simple sweat loss monitoring. FIG. 50 shows the features of amicrofluidic device for fluorometric sweat sensing. The diameter andtotal thickness of the device are 32 mm and ˜2 mm, respectively. Threeindependent assays are designed along the inside of the round layer eachwith its own inlet hole with diameter of 0.3-1.5 mm and connected threemicro-reservoirs, respectively. The channel widths and depths are100-200 μm and ˜400 μm, respectively, and the diameter of each reservoiris 2.64 mm. The micro-reservoirs are connected by the curved channelswith capillary bursting valves (CBVs). The valves enable time-sequentialsweat sampling with three intervals for each reservoir. FIG. 51 shows aset of CBVs in the chamber. CBV #1 has 50 μm wide channel with 123°, CBV#2 has 160 μm wide channel with 23°, CBV #3 has 150 μm wide channel with85° of diverging outlet, respectively. Sweat first burst CBV #2 andfills the chamber #1. Then, CBV #3 with highest BP blocks the sweatflow. After filling chamber #1, sweat burst CBV #3 and flows to the nextchambers. The assay reservoir having three assay chambers can store atotal of 8.1 μL of sweat, with ˜2 μL for each chamber. The two roundreservoirs located between the assay systems were designed for afluorescence reference system composed of ionic liquid and afluorescence dye.

The doughnut-shaped black PDMS with 200 μm thickness placed on top ofthe device work as a light shield to prevent photobleaching of thefluorescence reagents while collecting sweat. The low elastic module(˜145 kPa) and surface adhesion property of the PDMS allowed fordetachable adhesion between the PDMS films without any treatment. ThePDMS-PDMS adhesion could be detached easily by figures (FIG. 50(c)). Theflower-shaped channel designed in the center of the layer allows thedevice to indicate sweat loss for the fluorometric assays. The incomingsweat dissolves the water-soluble dye located near the inlet as it flowspast, thereby creating a visible, colored fluid with an easilyidentifiable filling front in the channel. Since the channel volume(˜8.1 μL) is designed to be almost equal to that of an assay system(˜8.1 μL), the channel system can indicate the amount of sweat fillingthe fluorometric assay reservoirs, which are normally shielded by theblack film. The low modulus and high elasticity (up to ˜200%) of PDMSenabled soft and flexible devices, which are suitable as a skin wearablesensing system. FIG. 50(d) shows deformation of a representative deviceby bending and twisting. The device exhibited excellent strengthproperties against various mechanical forces and distortions and can beapplied to the skin on any part of the human body.

Device Fabrication: Soft lithographic techniques yielded themicrofluidic silicon molds. Patterning photo-resist of KMPR 1010(Microchem, MA, USA) on 1 mm thick Si-wafer and deep reactive ionetching (STS Pegasus ICP-DRIE; SPTS Technologies, Newport, UnitedKingdom) generated a mold for microfluidic channel. Thin layer ofpoly(methylmethacrylate) (PMMA; Microchem, MA, USA) formed on the mold.Pouring 10:1 PDMS (Sylgard 184; Dow corning, MI, USA) mixed with whitesilicone dye (Silc Pig; Smooth-on, Inc., PA, USA) at 10% wt on the moldand spin coating at 150 rpm with baking at 150° C. for 30 min yielded a1 mm thick layer. All the chemical assays were located on the cured PDMSchannel. Mechanical punches were used to cut out a round-shaped patchand to create inlet holes for collecting sweat. A transparent PDMSmixture in a ratio of 10:1 (rubber base:cure) casted on a PMMA coatedflat wafer at 300 rpm and cured 150° C. for 30 min formed a uniformcover layer. Bonding the cover film to the white microfluidic channelfilm after placing fluorometric assays defined sealed microfluidicchannels and assay chambers. A tiny amount of PDMS (10:1) was applied onthe cover film before stacking on top of the channel layer, and thencured at 40° C. for 1 h. The process allowed efficient bonding of thestack without damage of the assay reagents. Casting a PDMS mixturecontaining a black silicone (Silc Pig; Smooth-on, Inc., PA, USA) in aratio of 10:1:1 (rubber base:cure:black silicone) at 200 rpm and curedat 150° C. for 30 min yielded a uniformly black elastic film. The blackcover film was placed on the top of the stack without any bonding agentsto yield a detachable light shield. A CO₂ laser (Universal LaserSystems, AZ, USA) cut a double-sided skin adhesive membrane (PC2723U;ScapaHealthcare, CT, USA) into a round shape with defined sweat inletholes. The adhesive membrane with matching inlet holes was bonded to thebottom surface of the PDMS device on one side and to the skin on theother side. Plasma treating the micro microfluidic layer with a coronagenerator (Electro-Technic Products, IL, USA) created hydrophilicsurfaces on the PDMS that allowed efficient bonding of the PDMS layerand the adhesive.

A smartphone-based fluorometric imaging system applicable for a sweatsensing device: A smartphone system yields fluorescence sweat sensingin-situ with microfluidic devices. FIG. 52(a) illustrates the featuresof a smartphone-based fluorescence imaging system consisting of a normalsmartphone attached an accessory. The attachment involving a dark shieldbox with immobilized excitation and emission filters allows a normalsmartphone to take fluorescence images using its camera function. Theattachment includes two movable parts: one is for fixing the holder tothe side of a smartphone, and the other is for adjusting the boxposition to make contact with the excitation and emission filters andthe interfaced smartphone LED light and camera (FIG. 52(b)). The filtersallowed the LED light and camera to work as an excitation light and adetector for fluorescence signals (FIG. 52(c)). The blue transparentfilms that are generally used for display enabled transmittance of onlyblue light with narrow wavelength (451±35 nm) (FIG. 52(d)) from thesmartphone LED light (400 nm-750 nm wavelength, FIG. 52(f)). Thetransmitted blue light allows the fluorescent probes (excitationwavelength of 400 nm-530 nm) on the patch to be excited. To detect onlythe emitted fluorescence signal, a long-wave pass glass lens that canblocks light below 515 nm wavelengths was placed at the interface of thesmartphone camera lens. Double green filters also provide green lightwith narrow wavelength (550±50 nm) from the smartphone LED light (FIG.52(e)). It means various excitation lights can be obtained from filteredsmartphone LED light.

FIG. 53(a) shows a procedure of fluorescence sweat sensing using themicrofluidic device and the smartphone-based system. The skin-mountedmicrofluidic device introduced sweat from the co-glands to theflower-shaped channel and the three independent assay parts through thecorresponding inlet holes (FIG. 53(a)-1). Completely filling theflower-shaped channel with the blue-colored sweat fluid indicates thatthe three assay reservoirs might be full. Then, the uppermost black filmcould be detached for taking a picture by the smartphone system (FIG.53(a)-2). Taking a picture with flashlight using the smartphone with theattachment provided a fluorescence image of the signals on the device(FIG. 53(a)-3). PDMS that is transparent for wide range of wavelengthand has a low refractive index (around 1.41) is applicable forfluorescence analysis.

The fluorescence signal intensity depends on the concentration oftargets. To calibrate the fluorescence signals, the intensity analyzedby Image J software (NIH, USA) was divided by the reference intensity(FIG. 53(b)). A stable fluorescence dye dissolved in ionic liquid waspreplaced as a reference in the device. The reference marker should havealmost same excitation wavelength to those of probes. Nonvolatile ionicliquid enabled placement of the reference dye in vapors permeate PDMSstably. Various fluorescence colored references are prepared by usingionic liquid and dyes.

In addition, a white sweat device played an important role forenhancement of the fluorescence signals due to reflection of emittingfluorescence by titanium oxide particles of white pigments on thecurvature of the micro reservoir (FIG. 54).

Device Fabrication: Assembly of black acrylic pieces (McMaster-Carr, IL,USA), excitation (Scotchcal™ graphic film, 3632-87; 3M, MN, USA)emission filters (colored-glass alternative filter, 5CGA-515, NewportCo., CA, USA), and a commercial smartphone holder (Lotus Tech, Wembley,UK) part using glue yielded a smartphone-based fluorometric-imagingdevice. The CO₂ laser cut an acrylic black board with 3.18 mm into eightpieces. Gluing the four black plates together formed a square shapedbox. Placing square plates with two holes for excitation and emissionfilters on the top of the box defined the light-shielding box. Theexcitation and emission filters were fixed to the holes of the plate.The box was attached to the smartphone holder by a long rectangularacrylic piece with a screw. For alignment of the sweat patch, a squareplate having a hole with a size equivalent to that of the patch wasplaced on the bottom of the box. Putting pieces of black paper on thesurface of the plates inside the box to prevent light reflectioncompleted the assembly process. All the results of the fluorescenceimages were taken by using a smartphone, iPhone 6 Plus (Apple Inc., CA,USA).

Reference marker: Dissolving 0.4 mg of rhodamine 110 chloride(Sigma-Aldrich, MO, USA) in 2 mL of 1-ethyl-3-methylilimidazolium ethylsulfate ionic liquid (Sigma-Aldrich, MO, USA) formed the green referencesolution. Drop casting 0.5 μL of the ionic liquid dye onto the chambersdesigned for the reference fluorometric dye completed the process.Dissolving 0.4 mg of rhodamine Red-X (Thermo Fisher, USA) in 2 mL of1-ethyl-3-methylilimidazolium ethyl sulfate ionic liquid formed the redreference solution.

Fluorometric development: Dropping assay solution onto respectivechambers of a microfluidic layer, and then drying at 35° C. for 1 h in alight shielded environment yields a solid-state fluorometric assay forvarious biomarkers. FIG. 55(a) shows the assay chambers for chloride,sodium, and zinc before and after filling sweat under visible light. Thefluorescence probes installed in each reservoir are easily dissolved byincoming sweat and reacted with their targets, chloride, sodium, andzinc selectivity. FIGS. 55(b), (c), and (d) show the variation of thefluorescence images of chloride, zinc, and sodium probes reacted withartificial sweat at pH 6 containing various concentration of the targetsunder the excitation light of the smartphone. The graphs below theimages show the dependence of the normalized intensity on theconcentrations of the targets. The standard curves worked forcalculating the concentration of the targets on a human trial. Thecalculated values were comparable with the values measured by thetraditional methods, ion chromatography for chloride, ICP-MS for zinc,and atomic absorption for sodium (FIG. 56). The fluorometric assay workseven in use of an extremely small amount of sweat. FIG. 57 shows theresult of fluorometric chloride assay using 0.3 μL artificial sweatcontaining with 0-150 mM chloride. Lucigenin was placed in amicrofluidic device using supporting papers.

Fluorometric assays: The chloride fluorometric assay solution consistsof 2 mg lucigenin (Sigma-Aldrich, MO, USA) dispersed in 1 mL of MilliQwater. The zinc fluorometric assay solution was prepared by adding 25 μLof zinc detector (Zinc Quantification Kit (Fluorometric), Abcam Inc.,MA, USA) into 5 mL of the zinc assay buffer. Dissolving 1 mg of thesodium detector (CoroNa™ Green; Molecular Probes, OR, USA) in 100 mL ofdimethyl sulfoxide (Sigma-Aldrich, MO, USA) yielded a concentratedsolution. Dispersing 2.3 μL of the concentrated solution into 1 mL ofMilliQ water yielded the sodium fluorometric assay solution at theconcentration of 40 μM. Dropping 2 μL volume of each assay solution ontothe respective chambers of the microfluidic layer, and then drying at35° C. for 1 h in a light shielded environment yielded the solid-statechloride, zinc, and sodium assays, respectively.

Example 13: Soft, Multi-Functional Microfluidic Device for ColorimetricAnalysis of Sweat Creatinine and Urea

A soft microfluidic device made from PDMS has flexibility and interfacesto the skin (FIGS. 58a and b ). The device provides severalfunctionalities that: 1) analyzes the concentration of creatinine, urea,and pH in sweat, 2) calculates instantaneous sweat rate and local sweatloss via adhesive layer that provides a water-tight sealing between skinand the device that enables the device to collect sweat continuously(FIG. 58c ). Sweat gland under open region of skin under adhesivegenerates sweat flow about 2 kPa to 1) inlet #1 fills the serpentinechannel then shows sweat rate and the local sweat loss, 2) inlet #2, 3and 4 fills the collection chambers in clockwise sequential mannerthrough the guide of series of capillary bursting valves and developcolor for the detection of pH, creatinine, and urea in sweat. Forcolorimetric analysis, each chamber has a chemical assay paper thatdevelop color according to target biomarker in sweat and color referencemarkers placed next the chamber to provide a standard color of targetbiomarker concentration for accurate color analysis that is not affectedby light condition. The exploded view of the device shows the detailedcompositions of one device (FIG. 58d ). The adhesive layer attaches thePDMS device on to the skin and the hole in the adhesive opens a routefor the sweat from the region to enter to the microfluidic channels.White microfluidic PDMS channel layer formed by soft lithography hasfour channels: the bottom serpentine channel for measuring sweat rateand local sweat loss, other circular chambers for measuring pH,creatinine, and urea concentration in sweat. The chemical assaycomponents are placed in each chambers and channel for their purpose. A200 μm thick clear 10:1 PDMS capping layer, treated with oxygen plasmato make it sticky, was placed onto microfluidic PDMS channel, togenerate the closed channel. On top the capping layer, a 25 μm thickthin PET film with reference color marker provide an accurate coloranalysis.

Device Fabrication: Fabrication begins with making a silicon wafer mold.Patterning photo-resist of KMPR 1010 (Microchem, MA, USA) on 1 mm thickSi-wafer and deep reactive ion etching (STS Pegasus ICP-DRIE; SPTSTechnologies, Newport, United Kingdom) generated a mold for microfluidicchannels and reservoirs. Thin layer of poly(methylmethacrylate) (PMMA;Microchem, MA, USA) formed on the mold as an anti-adhesion layer.Pouring 10:1 PDMS (Sylgard 184; Dow corning, MI, USA) mixed with whitesilicone dye (Reynolds Advanced Material, 5% wt) on the mold and spincoating at 200 rpm with baking at 70° C. for 45 min. Capping layer wasspin coated with 10:1 PDMS at 200 rpm and baked at 70° C. for 45 min.Both microfluidic channel layer and capping layer were treated withlaboratory corona treater (Electro-Technic Products) for better adhesionright before loading assays. All the chemical assays were located on thecured PDMS channel. 25 μm thick clear polyester film (THERMLfilm SELECT®10852; FLEXcon, MA, USA) on the top of the device with color referencemarker. 60 μm thick medical grade acrylate adhesive (1524; 3M, MN, USA)bonded to the bottom of the device with 30 sec of corona treatment.

Color development and reference marker: Colorimetric method fordetection of biomarkers needs a color reference marker for accurateanalysis of color regardless of light condition. FIG. 59a shows thecollection of color reference markers for analyzing pH, creatinine, andurea from sweat. For the preparation of the color reference marker, invitro test with standard solution produced reference color and digitalimaging and image analysis provides color value of each assay. From thevalues, the color reference marker is generated and printed on the thinand clear film and attached to the top of the device. Creatinine in thesweat produces hydrogen peroxide (H₂O₂) from the enzymatic reaction withcreatininase, creatinase, sarcosine oxidase, and peroxidase and reactswith probe using H₂O₂ results red color that changes green level in thechamber dominantly (FIG. 59b ). Urease immobilized in pH paperdecomposes urea in sweat into ammonia, changing the color of pH paperfrom yellow to green, where the red level changes dominantly with ureaconcentration (FIG. 59c ). Universal pH dye provides a pH sensor and redlevel from the sensor that changes dominantly with pH of solution servea comparing parameter of the color of assay (FIG. 59d ).

Colorimetric assay: 1) urea: 0.01 mg/ml urease solution was preparedwith urease (urease from Canavalia ensiformis, Jack bean, type III;Sigma-Aldrich, MO, USA) in deionized water. Urea assay paper wasprepared by immobilizing 2 μl of urease solution onto a pH paper(diameter, 3 mm, Hydrion Strips B 1-11, Micro Essential Laboratory, NY,USA) and drying under vacuum in a desiccator for 15 min.

2) creatinine: the creatinine assay solution was generated by fullymixing 24 μl of buffer, 8 μl of each enzyme solution, creatininase,creatinase, and enzyme mix, and 2 μl of probe. Creatinine assay paperwas prepared by spotting 2 μl of cocktail solution onto a filter paper(diameter, 3 mm) and drying under vacuum in a desiccator for 15 min(Creatinine Assay Kit; Sigma-Aldrich, MO, USA). A metal punch (diameter,3 mm) was used to create circular pH papers for urea and filter papersfor creatinine.

3) pH: pH cocktail solution was realized by thoroughly vortexing 4 ml ofuniversal pH dye (Fisher Scientific, NH, USA), 274 mg of polyvinylchloride (M.W. ˜233,000, Sigma-Aldrich, MO, USA), 635 μl ofo-nitrophenyloctylether (Sigma-Aldrich, MO, USA) and 508 μl of aliquotin 10 ml of tetrahydrofuran (Sigma-Aldrich, MO, USA) till a homogenoussuspension was obtained. Thereafter, a filter paper was dipped in thecocktail solution for 10 s and allowed to dry at ambient conditions for15 min to realize the solid-state pH assay. Finally, a metal punch(diameter, 3 mm) was used to excise circular pads of the pH assay paperfor incorporating in the wearable patch.

Standard color development and color reference marker preparation:Creatinine solution was prepared by dissolving creatinine fromcreatinine assay kit (Sigma-Aldrich, MO, USA) in DI water. Urea(Sigma-Aldrich, MO, USA) generated standard solutions in DI water as itsconcentrations. pH buffer solution was made by 1×PBS buffer (pH 7.4,Sigma-Aldrich, MO, USA) and hydrochloric acid (37%, Sigma-Aldrich, MO,USA), and pH meter (Mettler Toledo, Greifensee, Switzerland) measuredit. For creatinine, urea and pH test, pipetting 2 μl of standardsolution into the chambers. For full color development, the device withcreatinine and urea assay filled by the solution stayed in the oven at37° C. for 15 min and pH for 5 min. A digital SLR camera (EOS 6D; Canon,Tokyo, Japan) took the picture of the device. Photoshop (Adobe Systems,CA, USA) provided color extraction from the color in the chambers. Acolor laser printer (C454 PS; Konica Minolta, Tokyo, Japan) produced areference maker on PET film at 1200 DPI resolution. The printed thereference marker placed on the device again and smartphone camera(Iphone 5s; Apple, CA, USA) took picture of the chamber with referencemarker. The color analysis compared the color level from the chamber andreference marker. Three spots from each chamber and reference markerprovided the average color value. By adjusting brightness of the image,repetition of printing and comparing provided the optimum referencemarker.

Method to improve accuracy of colorimetric assay analysis: Accuracy ofcolorimetric assays suffer in response to effects such as subtle colorchanges between different states (e.g. difference between 5 mM and 10 mMchloride levels), non-uniform lighting conditions, channel height, orvariations in printed calibration marks (e.g. resolution, inkconcentration, color print space). Whereas sampling colors in adevice-independent color space, such as CIE L*a*b* color space, providesa facile method for color comparison, many colorimetric assays (such aschloride) contain a “white point measurement” indicating the absence ofan analyte (i.e. 0 mM chloride). The utilization of white in acolorimetric assay is problematic when attempting to distinguish betweensubtle variations in color and thus analyte concentration as white isdefined as L*=100, a*=0, b*=0. Luminance, L*, is most prone tovariations in illumination, which propagates uncertainty intocolorimetric assay analysis at low concentrations. For clinicalapplications, maximizing accuracy at low concentrations, which forchloride is <45 mM, is necessary to establish the assay as comparable toa diagnostic gold standard (e.g. chloride value <=1 mM standarddeviation).

A flatbed scanner (Canon CanoScan LiDE 220) is used to eliminatevariations in lighting. Illumination uniformity can be determined via apixel-by-pixel variation analysis of each channel (RGB) of a full-bedscan. Typical variation was found to be <0.8% across the entire bed with<0.1% variation across a 30 mm×60 mm region (size of a test sweatdevice).

One strategy to improve colorimetric assay accuracy is to eliminate thewhite point in an assay by overprinting the measurement region with acontrasting color. By overprinting, the relevant detection range of theassay is elongated to increase the range of distinguishable colormeasurements. A demonstration of this strategy appears in FIG. 60.

Although several methods exist to relate measured differences in colorin CIE L*a*b* space to assay concentration, accuracy is maximized whileminimizing external factors (such as luminance variation) by using theestablished value for chroma (C) which is related to the a* and b*coordinates via the relationship C=(a^(*2)+b^(*2))^((1/2)). Usingchroma, independent of L*, to map measured color to known analyteconcentration establishes a calibration curve to measure unknownsolutions. Identifying the optimal color for a particular colorimetricassay is rapidly ascertained via a facet plot of the different variablesso as to identify the parameters that provide a linear fit with the bestfit and the largest gradient (i.e. slope) as compared to the controlpoints. An example of the facet plot appears in FIG. 61 for the Greencolor shown in FIG. 60.

A comparison of color overlays generated via an inkjet printer and alaser printer show minimal influence on the performance of a selectedcolor.

After the identification of an optimized color and opacity for a givenassay, comparison of the calibration curve to an “unknown” calibrationsample provides a simple means for evaluating accuracy. For silverchloranilate assay for chloride samples in a diagnostically relevantrange (10 mM to 75 mM), the best-fit regression equation has beendetermined to be a power-law fit. The R² values for control is 0.995 andfor the green-color overlay 0.999 for the values provided in Table 3.Evaluating the calibration curve at the measured chroma values for 30 mM(not part of fit calculations), the control yields a concentrationmeasurement of 25.7 mM, within the expected range of a colorimetricassay. However, when measured with a color overlay, the fit yields aconcentration of 30.41 mM. The calibration solution, when measured usinga chloridometer (clinical gold standard, Wescor Chlorochek), is 30.5 mM(N=3, resolution is ±1 mM).

TABLE 3 Measured chroma values for a control (no overlay) and greenoverlay for silver chloranilate chloride assay at given chlorideconcentrations. 30 mM represents the calibration solution that is usedto check the goodness of fit and compare performance accuracy. ChromaConcentration (mM) Control Green Overlay 10  7.07 53.15 25 11.66 48.0950 20.62 40.02 75 25.81 33.12 Calibration: 30 12.81 46.20

Example 14: Integrating Structural Features for Rapid Volume Readout

The planar microfluidic channels can be designed so that the method offilling provides information about the performance of the device. Oneexample is the use of filling behavior in a spiral to indicate the fillpercentage of a channel “reservoir” of a known volume. As shown in FIG.62, a channel “reservoir” holds a total volume of 5 μL. The featurefills continuously but when half full (2.5 μL) the direction of fillingswitches. Using both motion and geometry, a person wearing the devicecan quickly monitor the collected sweat volume. As the sweat fills thedevice continuously, approximate percentages (such as ⅔ and ¾) are alsoeasily assessed. When combined into a series of reservoirs, largervolumes of sweat can be quickly measured during a collection period asshown in FIG. 63.

Example 15: Device Performance (U.S. App. No. 62/514,515)

To test device performance in a laboratory setting, we conducted a studyon a small (n=3) number of adult volunteers substituting our device forthe Macroduct® sweat collection system. The study assessed thecollection performance over a 9-day period with a variable hydrationstate (FIG. 64A), a contra-lateral study of the device efficacy vs.Macroduct device (FIG. 64B), and comparison of chloride values ofcollected sweat evaluated using the ChloroChek (FIG. 64C). For all casesno QNS instances occurred. Over the 9-day volume study, the collectiondevice demonstrated reproducible collection performance.

Our novel epifluidic device collected at least 40 μL of sweat regardlessof hydration state during the 30 min collection timeframe and for themajority of the study days sweat collected was in excess of 80 μL.Arm-to-arm variation was within expected ranges for iontophoreticstimulation (<35%). As shown in FIG. 64, an initial contra-lateral studybetween the Macroduct and the collection device on three adultvolunteers (same day) demonstrated performance equivalence. Variationobserved for Subjects 1 and 3 is within the expected aforementionedrange; however, the device collection volume for Subject 2 indicates thepotential for enhanced collection performance. This possibility will beinvestigated as part Specific Aim #1 of this proposed study.

Initial validation of the novel epifluidic collection device performancerequired verification of chloride level similarity between sweatobtained via the Macroduct® pilocarpine stimulation and collectiondevice. FIG. 64C shows the chloridometer (ChloroChek) measurements (N=5runs) of each sweat sample from a single iontophoresis stimulationsession (separate arms). The absence of variation between the measuredvalues for the two subjects indicates that both the teal dye ischloride-free and no difference exists between the sweat collected viaeither method (beyond standard biological variation).

Advanced Functionality: The ability to integrate additionalfunctionality beyond sweat collection offers a key advantage forepidermal microfluidic devices over the existing sweat collectionmethods. Reconfiguration of the epifluidic sweat collection device toinclude a colorimetric assay for the quantitative analysis of sweatchloride levels offers the possibility of significantly decreasing thetime-to-answer at the point-of-care for CF diagnostics or offering afacile method for an initial chloride level screen. FIG. 65A showcasesthis device variation which features an integrated colorimetric assayfor chloride and an independent sweat collection chamber (70 μL volume).The colorimetric assay uses an excess of silver chloranilate [12] forthe quantitative analysis of chloride levels in sweat [11]. Theintensity of the violet color (FIG. 65B) increases with increasing sweatchloride levels. Imaging the assay reservoirs with a smartphone cameraprovides a simple method for rapidly quantifying the reservoir color bycomparing the measured color with a calibrated color reference (notshown).

A small, one-person study of the sweat collection reproducibility of thedevice variation revealed lower volumes of collected sweat; however, noinstants of QNS were recorded. This decrease in collection volumereflects the elimination of two additional collection points to provideindependent colorimetric analysis of sweat chloride levels (FIG. 66A).FIG. 66B shows assay performance against the ChloroChek benchmark over a4-day trial. Although the colorimetric assay results indicate anelevated level of chloride, as compared to the measurements from achloridometer, the assay correctly indicates the absence of CF in theadult volunteer. As a screening method, this integration with aneffective collection device offers a promising opportunity for on-boardchloride screening.

Measurements of sweat loss and instantaneous sweat loss: The serpentinemicrofluidic channel has capability to measure sweat rate over a localregion (anterior forearm) during cycling, and to correlate this measurewith the total body sweat loss (FIG. 67a ). The simple microfluidicdevice with colored dye in the channel shows the filling of the sweatfrom the skin (FIG. 67b ). The comparison of the sweat collection fromthe microfluidic device with total body loss which measured by weighingbody weight before and after exercise without water consumption showsgood correlation (FIG. 67c ), indicating that microfluidic devices couldbe used to estimate total body loss in ambulatory environments. Theamount of sweat captured with the microfluidic device and the controlmethod using fabric based skin patch (Tegaderm® absorbent pad) alsoshows good correlation (FIG. 67d ). Furthermore, microfluidic devicesenable measurement of instantaneous sweat rates during exerciseroutines. FIG. 67e shows instantaneous sweat rates at three differentintervals in time. During the first session of exercise (labeled‘exercise’), there is constant sweat rate followed by a decrease insweat rate approaching zero sweating when the subject is at rest(labeled ‘rest’). The instantaneous sweat rate returns to initial levelsonce the subject re-initiates physical exertion (labeled ‘resumedexercise’).

Control sweat collection and Total body loss measurement: Subjectsperformed exercise on standing bicycle for 20-90 min with no fluidintake or restroom use during exercise. Tegaderm® with pad (3582; 3M,MN, USA) provided a control method to measure sweat generation atdefined region. After collecting the sweat from the skin, the sweatweight was calculated by subtracting the initial mass of the Tegaderm®.Weighing by digital scale (Adam Equipment, CT, USA) with 2 g accuracybefore and after exercise in nude provided the data for calculatingtotal body loss.

Field testing involves healthy, non-diabetic, human subject volunteers(three males) instrumented with devices on the upper wrist. The physicalexercise involves cycling on a stationary bike with increasingresistance. Real-time data acquisition during each trial occurs eitherthrough a compact, short-range reader, or an extended, long-range readerwere positioned in the vicinity of the device. The long-range readeroffers significant spatial latitude to the user during data collection.FIG. 68A displays an image of a subject on a stationary bike wearing thepatch, with an extended antenna (60×30 cm2) in the background. FIG. 68Bsummarizes the effective communication distance between the device andantenna (shown in FIG. 68A), presented here is the largest distance thatenables successful operation. The data shows a maximum operatingdistance of about 18 cm with this configuration.

FIG. 68(C-E) shows a device after cycling, along with a summary of dataacquired from the lactate and glucose sensors. Similarly, FIG. 68(F-H)presents images of the device for another subject, illustrating lactateand glucose sensor measurements. For both subjects, the respectiveelectrochemical sensors produce voltage signals that yield correspondingconcentrations based on calibration plots obtained at ˜300 C (usualsweat temperature). Analyte concentrations reported in these studies areconsistent with previously published studies 21,38. Image analysis ofFIGS. 68(C and F) reveals that the concentration of chloride is 34±2 mM(subject #1; chamber #1) and 62±5 mM (subject #2; chamber #1) and 36±5mM (subject #2; chamber #2); the pH is 6.4±0.1 (subject #1; chamber #1)and 6.3±0.1 (subject #2; chamber #1) and the sweat rate is ˜0.52 μl/min(subject #1) and ˜0.88 μl/min (subject #2). Separate analyses usingconventional techniques such as commercial bench-top chloridometry, pHanalysis and high-resolution nuclear magnetic resonance (NMR)spectroscopy provide points of comparison. FIG. 68I illustratescapabilities in monitoring of glucose and lactate levels in sweat overmultiple days for subject #1. See FIG. 69 for data from an additionaltwo subjects (subject #2 and #3). Separate measurements capture bloodlactate and glucose levels over the same time period, as points ofcomparison. In these studies, the subjects wear sensors on the upperwrist for two consecutive days. On each day, the subject performs acycling exercise on a stationary bike once in the morning in a fastingstate, 20 min after consuming a sweetened drink containing 150 g ofsugar and then again in the evening. Blood tests with commercial bloodlactate (Lactate Plus®, Nova Biomedical, MA) and blood glucose(Accu-Check® Nano meter, Roche Diabetes Care, Inc.) meters capture theconcentrations of these analytes before and after each cycling event.Photos of the device at different stages of the study, as shown in FIG.68I and FIG. 69, indicate robust adhesion to the skin throughout thestudy. Analysis of the data reveals that the blood levels after eachsession follow trends that are similar to those of data measured onsweat using the skin-interfaced devices. These findings are generally inagreement with those of previous studies that compare lactate andglucose levels in blood to those measured in sweat using conventionalcollection and ex-situ analysis techniques 44,70. Further support forthe long-term stability of the sensors follows from comparisons ofsignals produced by a pair of devices after the two-day trial with anunused pair (FIG. 70). The data show that the performance of the glucosesensor remains unchanged while the response of the lactate sensordecreases by only ˜20% even after these rigorous two-day trials. Theseresults represent the first examples of long-term use of skin-interfacedsweat sensors. The outcomes suggest potential for non-invasive trackingof blood glucose and lactate levels.

Example 16: Battery Free NFC Based Soft Microfluidics to Readout SweatExcretion Rate

Using the system packaged, human tests were conducted. FIG. 71a showsthe device can be mounted on various location of the subject body. Thehuman tests were conducted to show the difference of sweat rate in thethermal environment (FIG. 71b ) and exercise environment (FIG. 71c ).Also in the exercise test, two locations on the bodies were compared.Subject #1 and #2 were tested and both showed the chloride and sweatrate were higher at running condition. The correlation of sweat rate andchloride concentration was also allowed as reported by Smith et al. in2011 and Taylor et al. in 2013. Also other four subjects (FIG. 71f-i )were tested to see the difference of sweat rate for forehead andforearm. Forehead is known to be a location which has most dense sweatgland, and generally shows high sweat excretion rate and pressure. Thefour subjects show similar results and sweat excretion trend.

For human test, four healthy volunteers were involved in the on-bodytest with jogging and operating stationary bike in door with 00%humidity and 25° C. temperature condition. The devices were placed onfore head, chest, lower back, and fore arm. Before mounting the device,the skin was cleaned with 70% methyl alcohol. In the process of humantest, there was no drinking water for hydration.

A second set of studies focus on exploring temporal variations in sweatglucose and lactate as compared to those of blood due to consumption offood and engagement in physical exercise. Here, the subjects wear thepatch for one day and perform a cycling exercise (15-20 min) on astationary bike in the morning in a fasting state, 30 min and 90 minafter consuming breakfast and then again 30 min before lunch and 30 minand 90 min after lunch. Blood tests are performed using protocolssimilar to those for the first set of studies. FIG. 72 shows dataacquired for subject #1 and #2 during these long-term sweat monitoring.Analysis reveals that the blood levels after each session follow trendsthat are similar to those of data measured on sweat using theskin-interfaced devices. The sweat glucose values lag behind thoseacquired from blood tests by ˜30-60 min, while a much smaller time lagexists between blood lactate and sweat lactate. Such time lags isattributed to complex biological pathways through which the bloodconstituents reach other biofluids.

STATEMENTS REGARDING INCORPORATION BY REFERENCE AND VARIATIONS

All references throughout this application, for example patent documentsincluding issued or granted patents or equivalents; patent applicationpublications; and non-patent literature documents or other sourcematerial; are hereby incorporated by reference herein in theirentireties, as though individually incorporated by reference, to theextent each reference is at least partially not inconsistent with thedisclosure in this application (for example, a reference that ispartially inconsistent is incorporated by reference except for thepartially inconsistent portion of the reference).

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe invention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments, exemplary embodiments and optional features, modificationand variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by theappended claims. The specific embodiments provided herein are examplesof useful embodiments of the present invention and it will be apparentto one skilled in the art that the present invention may be carried outusing a large number of variations of the devices, device components,methods steps set forth in the present description. As will be obviousto one of skill in the art, methods and devices useful for the presentmethods can include a large number of optional composition andprocessing elements and steps.

When a group of substituents is disclosed herein, it is understood thatall individual members of that group and all subgroups, are disclosedseparately. When a Markush group or other grouping is used herein, allindividual members of the group and all combinations and subcombinationspossible of the group are intended to be individually included in thedisclosure.

Every formulation or combination of components described or exemplifiedherein can be used to practice the invention, unless otherwise stated.

Whenever a range is given in the specification, for example, atemperature range, a time range, or a composition or concentrationrange, all intermediate ranges and subranges, as well as all individualvalues included in the ranges given are intended to be included in thedisclosure. It will be understood that any subranges or individualvalues in a range or subrange that are included in the descriptionherein can be excluded from the claims herein.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art asof their publication or filing date and it is intended that thisinformation can be employed herein, if needed, to exclude specificembodiments that are in the prior art. For example, when composition ofmatter are claimed, it should be understood that compounds known andavailable in the art prior to Applicant's invention, including compoundsfor which an enabling disclosure is provided in the references citedherein, are not intended to be included in the composition of matterclaims herein.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. In each instanceherein any of the terms “comprising”, “consisting essentially of” and“consisting of” may be replaced with either of the other two terms. Theinvention illustratively described herein suitably may be practiced inthe absence of any element or elements, limitation or limitations whichis not specifically disclosed herein.

One of ordinary skill in the art will appreciate that startingmaterials, biological materials, reagents, synthetic methods,purification methods, analytical methods, assay methods, and biologicalmethods other than those specifically exemplified can be employed in thepractice of the invention without resort to undue experimentation. Allart-known functional equivalents, of any such materials and methods areintended to be included in this invention. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

We claim:
 1. A microfluidic system for monitoring a biofluid property,the system comprising: a flexible substrate; at least two microfluidicnetworks, each microfluidic network configured to independently monitora biofluid property; wherein each microfluidic network comprises: amicrofluidic inlet conduit network at least partially embedded in orsupported by the substrate; a biofluid inlet fluidically connected withthe microfluidic inlet conduit network to introduce a biofluid from askin surface to the microfluidic inlet conduit during use; a pluralityof reservoir chambers, each reservoir chamber fluidically connected withthe microfluidic inlet conduit network; and a plurality of capillaryburst valves fluidically connected with the microfluidic conduitnetwork, each capillary burst valve positioned between fluidicallyadjacent reservoir chambers.
 2. The microfluidic system of claim 1,further comprising a plurality of colorimetric sensors, wherein eachcolorimetric sensor is positioned in a unique reservoir chamber tomonitor the biofluid property.
 3. The microfluidic system of claim 1,wherein the at least two microfluidic networks differ from each otherby: (i) a biofluid inlet dimension, (ii) a reservoir chamber volume ofeach of the plurality of reservoir chambers, (iii) a burst pressure ofeach of the plurality of capillary burst valves, (iv) a chemicalcomposition of a chemically-mediated reaction chamber, or (v) anycombination thereof.
 4. The microfluidic system of claim 1, wherein afirst microfluidic network is configured to monitor a biofluid parameterassociated with a low-flow biofluid regime and a second microfluidicnetwork is configured to monitor a biofluid parameter associated with ahigh-flow biofluid regime, and the biofluid property is biofluid amount;biofluid analyte concentration; biomarker presence or absence; or acombination thereof.
 5. The microfluidic system of claim 4, wherein thehigh biofluid loss regime is at least 10 times greater than the lowbiofluid loss regime.
 6. The microfluidic system of claim 1, whereineach microfluidic network further comprises: at least one microfluidicoutlet conduit, each microfluidic outlet conduit fluidically connectedto at least one of the plurality of reservoir chambers and configured torelieve gas back pressure from the microfluidic inlet conduit network.7. The microfluidic system of claim 1, wherein the plurality ofreservoir chambers are chemically decoupled from each other forindependent biofluid property detection and/or time sequential biofluidproperty monitoring.
 8. The microfluidic system of claim 1, furthercomprising a biofluid gelling additive or an absorbent contained withinthe microfluidic network.
 9. The microfluidic system of claim 8, whereinthe biofluid gelling additive comprises two or more unique biofluidgelling additives.
 10. The microfluidic system of claim 8, wherein thebiofluid gelling agent is configured to mix or react with the biofluidto increase a biofluid viscosity.
 11. The microfluidic system of claim10, wherein, the increase in biofluid viscosity is by at least a factorof 2 of the biofluid viscosity before mixing or reacting with thebiofluid gelling agent.
 12. The microfluidic system of claim 8, whereinthe biofluid gelling agent comprises cellulose or a derivative thereof.13. The microfluidic system of claim 12, wherein the biofluid gellingagent is methyl cellulose or hydroxypropyl methylcellulose.
 14. Themicrofluidic system of claim 8, wherein the weight ratio of the biofluidgelling agent to biofluid, in at least one of the plurality ofreservoirs, is selected from the range of 0.1 to
 1. 15. The microfluidicsystem of claim 1, further comprising an expunge port fluidicallyconnected with the reservoir chamber for the removal of biofluid fromthe reservoir chamber.
 16. The microfluidic system of claim 1, whereinsaid flexible substrate comprises a material selected from the groupconsisting of polydimethylsiloxane (PDMS), polyurethane, cellulosepaper, cellulose sponge, polyurethane sponge, polyvinyl alcohol sponge,silicone sponge, polystyrene, polyimide, SU-8, wax, olefin copolymer,polymethyl methacrylate (PMMA), polycarbonate, polyvinyl chloride,chitosan, and any combination thereof.
 17. The microfluidic system ofclaim 1, further comprising an adhesive layer configured to mount thesystem to a skin surface.
 18. The microfluidic system of claim 17,wherein the adhesive layer reversibly adheres the system to the skinsurface.
 19. The microfluidic system or claim 18, wherein the adhesivelayer comprises medical grade acrylic or medical grade silicon.
 20. Themicrofluidic system of claim 1, further comprising a protective layerembedded in or supported by the flexible substrate.
 21. The microfluidicsystem of claim 20, wherein the protective layer prevents biofluid fromescaping from the reservoir chamber or the sweat sensor.
 22. Themicrofluidic system of claim 20, wherein said protective layer ispolyethylene.
 23. The microfluidic system of claim 1 that isincorporated into a glove.
 24. The microfluidic system of claim 1,wherein the biofluid property is visually observable.
 25. Themicrofluidic system of claim 1, wherein a signal corresponding to thebiofluid property is transmitted from said system to an externalreceiving device.
 26. The microfluidic system of claim 1, wherein thebiofluid property is one or more of sweat volume, sweat rate, or sweatloss.
 27. The microfluidic system of claim 1, wherein the biofluidproperty is pH.
 28. The microfluidic system of claim 1, wherein thebiofluid property comprises the presence of, amount or concentration ofan analyte in said biofluid or component thereof.
 29. The microfluidicsystem of claim 28, wherein said analyte is an electrolyte, ametabolite, or a biomarker in said biofluid or component thereof. 30.The microfluidic system of claim 1, wherein a leading edge of biofluidin a sensor microfluidic channel or reservoir is sensed as a function oftime.
 31. The microfluidic system of claim 30, wherein the leading edgeis sensed visually or measured using a photodetector.
 32. Themicrofluidic system of claim 1, wherein the flexible substrate is afunctional substrate.
 33. The microfluidic system of claim 1, furthercomprising an electronic sensor operably connected to the microfluidicnetwork, wherein an amount of biofluid is proportional to an electricalresistivity or electrical conductivity parameter measured by the sensor.34. The microfluidic system of claim 1, comprising a disposable portioncomprising the microfluidics network and a reusable portioncorresponding to an electronic device, wherein the disposable andreusable portions are connected to each other by one or more selectivelyreleasable coupling elements.
 35. The microfluidic system of claim 34,wherein the selectively releasable coupling elements comprise a magnet.36. A method of analyzing biofluid from a subject, the method comprisingthe steps of: contacting the flexible substrate of claim 1 with a skinsurface of a subject; and analyzing the biofluid from the skin surface.37. The method of claim 36, wherein said biofluid is sweat.
 38. Themethod of claim 36, wherein said subject is a human subject undergoing adiagnostic procedure, a therapeutic procedure, or a fitness activity.39. The method of claim 36, wherein said subject is a human subjectmonitoring the presence, onset or progression of a disease condition.40. The method of claim 36, further comprising the step of increasingbiofluid retention in the system by one or more of: increasing biofluidviscosity in the microfluidic network; and/or absorbing biofluid to anabsorbent.
 41. The method of claim 36, wherein the analyzing comprises:observing biofluid volume in at least a portion of the microfluidicnetwork; and/or observing a colorimetric change in a reservoir chamber.42. The method of claim 36, wherein the contacting comprises conformallycontacting the flexible substrate with the skin surface.
 43. Themicrofluidic system of claim 1, further comprising a capping layerconnected to a skin-facing surface and/or a back-facing surface of theflexible substrate.
 44. A microfluidic system for measuring a biofluidpressure, the system comprising: a flexible substrate; a plurality ofreservoir networks at least partially embedded in or supported by theflexible substrate, wherein each reservoir network comprises: areservoir chamber; a biofluid inlet fluidically connected to thereservoir chamber via a capillary burst valve to introduce a biofluidfrom a skin surface to the reservoir chamber, wherein the capillaryburst valve has a burst pressure; and an outlet fluidically connected tothe reservoir chamber; wherein the burst pressure of each capillaryburst valve is selected to correspond to a pressure range of thebiofluid from the skin surface.
 45. The system of claim 44, wherein atleast one of the plurality of reservoir networks has a unique capillaryburst valve pressure.
 46. The system of claim 44, wherein the biofluidinlet is fluidically aligned with a biofluid source of the skin surfaceduring use.
 47. The system of claim 44, wherein each reservoir networkfurther comprises at least one colorimetric sensor to provide an opticalreadout.
 48. The system of claim 44, wherein at least a portion of thecapillary burst valves are fluidically aligned in a serial configurationand have a burst valve pressure that increases and spans a range that isgreater than 0 kPa and less than 10 kPa.
 49. The microfluidic system ofclaim 44, further comprising a biofluid gelling additive or an absorbentcontained within the microfluidic network.
 50. The microfluidic systemof claim 44, further comprising an expunge port fluidically connectedwith the reservoir chamber for the removal of biofluid from thereservoir chamber.
 51. The microfluidic system of claim 44, wherein saidflexible substrate comprises a material selected from the groupconsisting of polydimethylsiloxane (PDMS), polyurethane, cellulosepaper, cellulose sponge, polyurethane sponge, polyvinyl alcohol sponge,silicone sponge, polystyrene, polyimide, SU-8, wax, olefin copolymer,polymethyl methacrylate (PMMA), polycarbonate, polyvinyl chloride,chitosan, and any combination thereof.
 52. The microfluidic system ofclaim 44, further comprising an adhesive layer configured to mount thesystem to a skin surface.
 53. The microfluidic system of claim 44,further comprising a protective layer embedded in or supported by theflexible substrate.
 54. The microfluidic system of claim 44 that isincorporated into a glove.
 55. The microfluidic system of claim 44,wherein the biofluid property is visually observable.
 56. Themicrofluidic system of claim 44, wherein a signal corresponding to thebiofluid property is transmitted from said system to an externalreceiving device.
 57. The microfluidic system of claim 44, wherein thebiofluid property is one or more of sweat volume, sweat rate, or sweatloss.
 58. The microfluidic system of claim 44, wherein the biofluidproperty is pH.
 59. The microfluidic system of claim 44, wherein thebiofluid property comprises the presence of, amount or concentration ofan analyte in said biofluid or component thereof.
 60. The microfluidicsystem of claim 44, wherein a leading edge of biofluid in a sensormicrofluidic channel or reservoir is sensed as a function of time. 61.The microfluidic system of claim 44, wherein the flexible substrate is afunctional substrate.
 62. The microfluidic system of claim 44, furthercomprising an electronic sensor operably connected to the microfluidicnetwork, wherein an amount of biofluid is proportional to an electricalresistivity or electrical conductivity parameter measured by the sensor.63. The microfluidic system of claim 44, further comprising a disposableportion comprising the microfluidics network and a reusable portioncorresponding to an electronic device, wherein the disposable andreusable portions are connected to each other by one or more selectivelyreleasable coupling elements.
 64. The microfluidic system of claim 44,further comprising a plurality of distinct component layers arranged ina stacked configuration.
 65. The microfluidic system of claim 44,further comprising a capping layer connected to a skin-facing surfaceand/or a back-facing surface of the flexible substrate.
 66. Themicrofluidic system of claim 44, further comprising a plurality ofdistinct component layers arranged in a stacked configuration.
 67. Amethod of analyzing biofluid from a subject, the method comprising thesteps of: contacting the flexible substrate of claim 44 with a skinsurface of a subject; and analyzing the biofluid from the skin surface.68. The method of claim 67, wherein said biofluid is sweat.
 69. Themethod of claim 67, wherein said subject is a human subject undergoing adiagnostic procedure, a therapeutic procedure, or a fitness activity.70. The method of claim 67, wherein said subject is a human subjectmonitoring the presence, onset or progression of a disease condition.71. The method of claim 67, further comprising the step of increasingbiofluid retention in the system by one or more of: increasing biofluidviscosity in the microfluidic network; and/or absorbing biofluid to anabsorbent.
 72. The method of claim 67, wherein the analyzing comprises:observing biofluid volume in at least a portion of the microfluidicnetwork; and/or observing a colorimetric change in a reservoir chamber.73. The method of claim 67, wherein the contacting comprises conformallycontacting the flexible substrate with the skin surface.
 74. Amicrofluidic system to measure a biofluid property, the systemcomprising: a flexible substrate; a microfluidic inlet conduit networkat least partially embedded in or supported by the flexible substrate; abiofluid inlet fluidically connected to the microfluidic inlet conduitnetwork to introduce a biofluid from the skin surface to themicrofluidic inlet conduit during use; a plurality of reservoirchambers, each reservoir chamber fluidically connected with themicrofluidic inlet conduit network; a plurality of capillary burstvalves fluidically connected with the microfluidic conduit network, eachcapillary burst valve positioned between fluidically adjacent reservoirchambers; a microfluidic outlet conduit network fluidically connectedwith the plurality of reservoir chambers and configured to relieve gasback pressure from the microfluidic inlet conduit network; and aplurality of colorimetric sensors, each positioned in a unique reservoirchamber to measure a biofluid property; and wherein: (i) at least one ofthe colorimetric sensors has a color-responsive reagent to measurechloride.
 75. The microfluidic system of claim 74, further comprising acolor indicator strip positioned between any two fluidically adjacentreservoir chambers.
 76. The microfluidic system of claim 74, wherein atleast one of the colorimetric sensors has a color-responsive reagentcomprising silver chloranilate.
 77. The microfluidic system of claim 74,wherein the colorimetric sensors are configured to measure concentrationof chloride in the biofluid.
 78. The microfluidic system of claim 74,further comprising a capping layer connected to a skin-facing surfaceand/or a back-facing surface of the flexible substrate.
 79. Themicrofluidic system of claim 74, comprising a plurality of distinctcomponent layers arranged in a stacked configuration.
 80. Themicrofluidic system of claim 74, further comprising a biofluid gellingadditive or an absorbent contained within the microfluidic network. 81.The microfluidic system of claim 74, further comprising an expunge portfluidically connected with the reservoir chamber for the removal ofbiofluid from the reservoir chamber.
 82. The microfluidic system ofclaim 74, further comprising an adhesive layer configured to mount thesystem to a skin surface.
 83. The microfluidic system of claim 74,further comprising a protective layer embedded in or supported by theflexible substrate.
 84. The microfluidic system of claim 74, wherein asignal corresponding to the biofluid property is transmitted from saidsystem to an external receiving device.
 85. The microfluidic system ofclaim 74, wherein a leading edge of biofluid in a sensor microfluidicchannel or reservoir is sensed as a function of time.
 86. Themicrofluidic system of claim 74, wherein the flexible substrate is afunctional substrate.
 87. The microfluidic system of claim 74, furthercomprising an electronic sensor operably connected to the microfluidicnetwork, wherein an amount of biofluid is proportional to an electricalresistivity or electrical conductivity parameter measured by the sensor.88. The microfluidic system of claim 74, further comprising a disposableportion comprising the microfluidics network and a reusable portioncorresponding to an electronic device, wherein the disposable andreusable portions are connected to each other by one or more selectivelyreleasable coupling elements.